Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Melatonin is hormone regulated by the light/dark cycle. It is considered to be a signaling pathway of the circadian clock located in the hypothalamic suprachiasmatic nucleus. Melatonin also regulates functions involving the energetic and behavioral metabolism of mammals. However, the melatonin synthesis can suffer influence from other hormones, like insulin and other glucocorticoids, in pathological conditions and stress. Studies have shown that glucocorticoids seem to modulate clock genes in peripheral tissues leading to metabolic diseases. The objective of the present study was to evaluate the functionality of the pineal gland and clock genes modulation in rats treated chronically with dexamethasone. Male Wistar rats (200-250g) were divided in to two groups: Control (CON) and Dexamethasone-treated (DEX). Animals from DEX group had 2mg/kg body weigh of dexamethasone administered intraperitoneally for 10 consecutive days and control animals received equal volume of saline solution also intraperitoneally. The circadian profile of blood glucose was evaluated and presented increased points of hyperglycemia during the night (p<0,05). The animals were sacrificed at each time point and had their pineal gland and plasma collected. The obtained data demonstrate a significant hyperinsulinemia in DEX animals (p<0,05). The findings show a reduction in the melationin content (p<0,05) associated with a decrease in the enzymatic activity of AANAT (p<0,05). Also, in these points, DEX animals presented a reduction in the glucose transporter (Glut1) in the pineal gland and insulin receptor (Insr) (p<0,05). The levels of mRNA of the clock genes Bmal1, Per1, Per2, Cry1, Cry2 and Rev-erbα in the isolated pineal glands demonstrated an increase in the gene expression (p<0,05). These results suggest that chronic treatment with dexamethasone is capable of modulating melatonin synthesis, as well as the clock genes. This evidence also suggests the possible presence of a responsive element to the functional glucocorticoid in the promoter region of these genes in the pineal gland. / A melatonina é um hormônio regulado pelo ciclo claro/escuro. Sendo considerado um sinal de saída do relógio circadiano localizado no núcleo supraquiasmático do hipotálamo. A melatonina regula funções envolvendo o metabolismo energético e comportamental em mamíferos. A síntese de melatonina pode ser influenciada por outros hormônios, a exemplo da insulina e dos glicocorticoides em condições patológicas e estresse. Estudos têm demonstrado que os glicocorticoides parecem modular os genes relógio em tecido periférico ocasionando doenças metabólicas. O presente estudo teve como objetivo avaliar a funcionalidade da glândula pineal e a modulação dos genes relógio em ratos tratados cronicamente com dexametasona. Ratos Wistar (200-250g) foram divididos em 2 grupos: Controle (CON) e Tratados com dexametasona (DEX). Os animais DEX receberam 2mg/kg de peso de dexametasona intraperitonial durante 10 dias consecutivos e os animais CON receberam o volume correspondente de solução salina intraperitonialmente. Os animais de ambos os grupos tiveram o perfil de glicose avaliado e apresentaram pontos de hiperglicemia acentuada durante a noite (p<0,05). Os animais de ambos os grupos foram sacrificados nos ZT17 ao ZT22 e tiveram a glândula pineal e o plasma coletados. Os dados obtidos mostram um quadro de hiperinsulinemia nos animais DEX (p<0,05). Os animais do grupo DEX apresentaram uma redução no conteúdo de melatonina total (p<0,05) associada à diminuição da atividade enzimática da AANAT (p<0,05). Nesses mesmos pontos os animais DEX apresentaram uma redução do transportador de glicose (Glut1) na glândula pineal e receptor de insulina (Insr) (p<0,05). Os níveis de RNAm dos genes do relógio Bmal1, Per1, Per2, Cry1, Cry2 e Rev-erbα em pineais isoladas apresentaram aumento da expressão gênica (p<0,05). Estes resultados sugerem que o tratamento crônico com dexametasona é capaz de modular a síntese de melatonina, bem com também os genes relógio, estas evidências sugerem a possível presença de um elemento responsivo ao glicocorticoide funcional na região promotora desses genes na glândula pineal.

Orientador: Jose Roberto Bosqueiro / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T18:48:58Z (GMT). No. of bitstreams: 1 Rafacho_Alex_D.pdf: 17852608 bytes, checksum: ee7b970220681b508fc5519d53e49655 (MD5) Previous issue date: 2008 / Resumo: A resistência à insulina (RI) é uma condição que exige maiores níveis de insulina circulante e que normalmente são providenciados pelo aumento da função e população de células ß. A RI pode ser observada a partir de diversos modelos experimentais em roedores tais como os modelos transgênicos, de gravidez, submetidos às dietas hiperlipídicas e hipercalóricas e a partir de infusão venosa de glicose. Estes modelos têm sido úteis para a compreensão dos mecanismos compensatórios observados durante a RI. Os glicocorticóides são amplamente utilizados na indução farmacológica da RI em modelos animais e em seres humanos, com fins científicos. A ativação da sinalização da insulina e das proteínas reguladoras do ciclo celular é crucial para a função e crescimento das células ß adultas. No presente trabalho, apresentamos modelos para investigação da função e crescimento de células ß pancreáticas in vivo a partir da administração diária de três concentrações distintas de dexametasona (DEX) (0,1, 0,5 e 1,0 mg/kg, peso corpóreo, intraperitoneal - DEX 0.1, DEX 0.5 e DEX 1.0, respectivamente) por 5 dias consecutivos. A sensibilidade periférica à glicose e à insulina, parâmetros de secreção de insulina e histomorfométricos foram investigados. A análise dos níveis de proteínas relacionados à função e crescimento de células ß foi realizada por Western blotting. O tratamento com DEX induziu RI de maneira dose- dependente. Aumento da secreção de insulina em resposta à glicose foi observado tanto in vivo quanto ex vivo nos três grupos tratados com DEX. Ratos DEX 1.0, que apresentam hiperglicemia moderada e marcante hiperinsulinemia, exibiram aumento de 5,1 vezes na proliferação além de hipertrofia de células ß, com aumento significativo na massa de células ß comparado aos ratos CTL. Os ratos DEX 0.5, hiperinsulinêmicos, porém normoglicêmicos, também apresentaram aumento significante de 3,6 vezes na proliferação e modesta hipertrofia de células ß. Entretanto, os ratos DEX 0.1, que desenvolveram o menor grau de RI, compensaram à demanda de insulina apenas com aumento da função de células ß. Nenhuma alteração da freqüência de morte celular foi observada nas células ß dos três grupos DEX comparados ao grupo CTL. Foi observada ativação da via IRS-2/PI3- K/Akt/p70S6K, bem como da proteína retinoblastoma nas ilhotas do grupo DEX 1.0 e, em menor grau, no grupo DEX 0.5 quando comparados com as ilhotas do grupo CTL. Assim, aumentando a concentração de dexametasona induzem-se três graus de requerimento de insulina in vivo, servindo como modelo para investigação de alterações compensatórias em células ß. O aumento da demanda de insulina é compensado por aumento da função das células ß (em todos os GRUPOS DEX) e por hiperplasia e hipertrofia de células ß nos GRUPOS DEX 1.0 e DEX 0.5. Baseado nos presentes resultados concluímos que o aumento dos níveis circulantes de insulina parece ser o maior estímulo para proliferação e hipertrofia das células de células ß observado na RI induzida pela dexametasona. / Abstract: Insulin resistance (IR) is a condition that demand increased levels of circulating insulin that are normally provided by increase of ß-cell function and mass. The IR can be observed in several experimental rodent models such as transgenic, pregnancy, high-fat or high-caloric diet and from glucose infusion model. These models have aided in elucidating the compensatory mechanisms observed during the IR. The glucocorticoids are widely used to induce the pharmacological IR in animal models and in humans, with scientific purpose. Activation of insulin signaling and cell cycle proteins are crucial to the function and growth of adult ß-cells. At the present study, we showed models to investigation of pancreatic ß-cell function and growth in vivo from the daily administration of three different dexamethasone (DEX) concentration (0.1, 0.5 e 1.0 mg/kg, body weight, intraperitoneal - DEX 0.1, DEX 0.5 and DEX 1.0, respectively) for 5 consecutive days. The peripheral sensibility to glucose and insulin, insulin secretion and histomorphometrical parameters were investigated. The analyses of proteins related to ß -cell function and growth were done by Western blotting. DEX treatment induced IR in a dose-dependent manner. Incease of glucose-stimulated insulin secretion was observed in vivo as well as ex vivo in the three DEX groups. DEX 1.0 rats, that present moderate hyperglicemya and marked hyperinsulinemia, ehibited a 5.1-fold increase in ß-cell proliferation besides hypertrophy, with significant increase of ß -cell mass compared to CTL rats. DEX 0.5 rats, that are hiperinsulinemic and normoglicemic, also exhibited a significant 3.6-fold increase in ß-cell proliferation as well as ß -cell hypertophy. However, DEX 0.1 rats, which exhibited the lowest degree of insulin resistance, compensate for insulin demand by improving only ß -cell function. No alteration in cell death frequency was noted in ß -cells from the three DEX groups compared to CTL group. Activation of IRS-2/PI3-K/Akt/p70S6K pathway as well as the retinoblastoma protein in islets from DEX 1.0 and, in lesser extend, in DEX 0.5 group was observed compared to islets from CTL group. Therefore, increasing doses of dexamethasone induce three different degrees of insulin requirement in living rats, serving as a model to investigate compensatory beta-cell alterations. The increased insulin demand is compensated by increase of ß-cell function (in all DEX groups) and ß -cell hyperplasia and hypertrophy in DEX 0.5 and DEX 1.0 groups. Based on the present results we concluded that the augmented levels of circulating insulin seem to be the major stimulus for ß-cell proliferation and hypertrophy observed in dexamethasone-induced insulin resistance. / Doutorado / Fisiologia / Doutor em Biologia Funcional e Molecular

Introdução: Estima-se que 10% dos doentes com asma t€m sintomas e limita‚es importantes, como exacerba‚es freqƒentes ou redu„o persistente da fun„o respirat…ria As altera‚es do par€nquima pulmonar distal tem sido recentemente abordadas na fisiopatologia da asma. Apesar do uso de corticoster…ides, pacientes com asma refrat†ria t€m mais estresse oxidativo, assim como apresentam ativaao da iNOS. Al‡m disso, muitos dos dispositivos utilizados para administra„o de ester…ides inalat…rios geram partˆculas que n„o chegam efetivamente ‰s vias a‡reas distais e ao par€nquima pulmonar. Objetivos: Avaliamos os efeitos do tratamento com montelucaste ou dexametasona tratamentos associados ou n„o a um inibidor especˆfio da iNOS (1400W) na resposta eosinofˆlica, remodelamento da matriz extracelular, estresse oxidativo, conteŠdo de actina, c‡lulas positivas para IL4, IL5, MMP9, TIMP1, IFN, TGF do par€nquima em cobaias com inflama„o crnica pulmonar. Métodos: As cobaias foram inaladas com ovalbumina (grupo OVA) 2X/semana por 4semanas. Ap…s a 4Ž inala„o, as cobaias foram tratadas diariamente com montelucaste (grupo OVAM 10mg/Kg/PO/dia) ou dexametasona (grupo OVAD 5mg/Kg/IP/dia). O inibidor da iNOS, 1400W (grupo OVAW 1mg/kg/dia) foi administrado intraperitonealmente nos Šltimos 4 dias (OVAW, OVADW e grupos OVAMW). Ap…s 72 horas da 7Ž inala„o, as cobaias foram anestesiadas, e os fragmentos de tecido pulmonar distal foram submetidos ‰ avalia„o histopatol…gica. Resultados: Houve um aumento no infiltrado eosinofˆlco, nas c‡lulas positivas para IL4, IL5, TIMP1, MMP9, iNOS, IFN TGF, conteŠdo de actina, isoprostano PGF2 alfa, fibras col†genas e el†sticas nos animais OVA em compara„o com animais SAL (p<0,05). Houve uma diminui„o no nŠmero de eosin…filos, c‡lulas positivas para IL4, IL5, MMP9, TIMP1, IFN, TGF, conteŠdo de actina, col†geno e isoprostano PGF2 alfa em todos os grupos tratados em compara„o com animais OVA (p<0,05). O conteŠdo de fibras el†sticas foram reduzidas somente nos grupos OVAMW, OVADW e OVAW em compara„o com animais OVA (p<0,05). A associa„o de 1400W e o tratamento com montelucaste (grupo OVAMW) potencializou a redu„o do conteŠdo de actina, fibras el†sticas, isoprostano PGF2 alfa de c‡lulas positivas para IL4, IL5, TIMP1, IFN TGF e iNOS em rela„o ao grupo montelucaste (OVAM) (p<0,05). Os tratamentos com 1400W e dexametasona (grupo OVADW) contribuˆram para uma maior redu„o do conteŠdo das fibras el†sticas, actina e isoprostanoPGF2 alfa e o nŠmero de c‡lulas positivas para IL4, IL5, IFN e TIMP1 em rela„o ao grupo dexametasona (OVAD) (p<0,05). Conclusões: O tratamento com corticoster…ides associados ‰ inibi„o da iNOS contribuiu para uma maior redu„o da remodela„o da matriz extracelular, diminuiu o estresse oxidativo, e tamb‡m foi eficiente para atenuar a resposta inflamat…ria Th2 no par€nquima pulmonar distal. Por outro lado, o tratamento com montelucaste associado à inibição da iNOS mostrou uma maior eficácia para reduzir o teor de fibras elásticas, a ativação do estresse oxidativo, conteúdo de actina e expressão das células positivas para IL4, IL5 no parênquima pulmonar distal. Estas associações podem representar futuras ferramentas farmacológicas para o controle das alterações histopatológicas pulmonares distais induzidas pela inflamação crônica / Introduction: It is estimated that 10% of asthma patients have symptoms and important limitations such as frequent exacerbations or persistent reduction of resiratory function, despite the use of corticosteroids. The alterations of distal lung parenchyma have been recently evaluated on asthma pathophysiology, particulary in patients with refractory asthma and difficcult to control. These patients have increased oxidative stress responses, mainly with significant activation of iNOS. Aims: We evaluated the effects of montelukast or dexamethasone treatments associated or not to an iNOS inhibitor (1400W) on eosinophilic response, extracellular matrix remodeling, oxidative stress, actin content, IL4, IL5, MMP9, TIMP1, IFN gama, TGF beta positive cells of distal lung parenchyma in guinea pigs with chronic alergic inflammation. Methods: Guinea Pigs were inhaled with ovalbumin (OVA group) twice a week for four weeks. After 4th inhalation, GP were treated with montelukast (OVAM group-10mg/Kg/PO/day) or dexamethasone (OVAD group-5mg/Kg/IP/day). The treatment with iNOS inhibitor 1400W (OVAW group-1mg/kg/day) was given daily in the last 4 days (OVAW, OVADW and OVAMW groups). After 72 hours of 7th inhalation, GP were anesthetized, lung strips were retired and submitted to histopathological evaluation. Results: There was an increase in eosinophilic infiltrate, in the number of positive cells for IL4, IL5, TIMP1, MMP9, iNOS, IFN gama TGF beta, actin, isoprostane PGF2 alpha, elastic and collagen fiber contents in OVA animals comparing to SAL group (p<0,05). There was a decrease in the number of eosinophils, IL4, IL5, MMP9, TIMP1, IFN gama, TGF beta positive cells, collagen, actin and isoprostane PGF2 alpha content in all treated groups compared to OVA animals (p<0.05), but the treatment with montelukast did not reduce the positive cells for IFN gama, compared to OVA (p>0.05). Elastic fiber content were reduced only in OVAMW, OVADW and OVAW groups compared to OVA animals (p<0.05). The association of 1400W and montelukast treatments potentiated the reduction of actin, elastic fibres and isoprostane PGF2 alpha contents and the number of IL4, IL5, TIMP1, IFN gama, TGF beta and iNOS positive cells compared to montelukast group (p<0.05). The treatments with 1400W and dexamethasone contributed to a greater reduction of elastic fibers, actin and isoprostane PGF2 alpha contents and the number of IL4, IL5, IFNgama and TIMP1 positive cells compared to dexamethasone group (p<0.05). Conclusions: Corticosteroid treatment associated to iNOS inhibition contributes to a greater reduction of extracellular matrix remodeling, decreases the oxidative stress, and also is efficient to attenuate the Th2 inflammatory response in distal lung parenchyma. On the other hand, montelukast treatment associated to iNOS inhibition showed a higher efficacy to reduce elastic fibres content, oxidative stress activation, actin content and IL4 and IL5 expression in distal lung parenchyma. These associations may represent future pharmacological tools for controlling distal pulmonary histopathological alterations induced by chronic inflammation

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-27T13:42:37Z No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 4559464 bytes, checksum: 43cecca924dfc2d1ba9044bdebbad60d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-06-28T13:01:49Z (GMT) No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 4559464 bytes, checksum: 43cecca924dfc2d1ba9044bdebbad60d (MD5) / Made available in DSpace on 2016-06-28T13:01:49Z (GMT). No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 4559464 bytes, checksum: 43cecca924dfc2d1ba9044bdebbad60d (MD5) Previous issue date: 2012-05-21 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introdução: Dentre os principais mecanismos que causam a osteoporose está o uso de glicocorticoides. Os glicocorticóides induzem a apoptose nos osteoblastos e levar a diminuição do volume ósseo total. Diversos produtos são usados no tratamento da osteoporose e entre estes, alguns produtos naturais, tais como os fitoestrógenos. Estudos com o extrato de Ginkgo biloba (EGb) demonstraram efeitos promissores no tratamento da osteoporose. Este trabalho se propõe avaliar os efeitos do EGb sobre a apoptose de osteoblastos, densidade mineral óssea e na estrutura óssea de ratas Wistar com osteoporose induzida por glicocorticóide. Métodos: 60 ratas foram distribuídas em 5 grupos: controle, osteoporose, alendronato (alendronato de sódio - 0,2mg/Kg/dia), EGb1 (28 mg EGb/Kg/dia ) e EGb2 (56 mg EGb/Kg/dia). A osteoporose foi induzida através de injeções intramusculares de dexametasona. A fosfatase alcalina óssea foi avaliada no soro e a análise imunohistoquímica das proteínas Bcl-2 e Bax foram avaliadas nos cortes histológicos de mandíbula e fêmur. Na tíbia foram avaliadas a densidade mineral óssea, densidade radiográfica, densidade mineral cortical e parâmetros biomecânicos. O grupo controle foi comparado ao grupo osteoporose (teste “t” Student/Mann Whitney). Os demais grupos, exceto o controle, foram analisados através do teste de ANOVA seguido do teste post hoc de Tukey ou Dunnett T3 (p<0,05). Resultados: No grupo osteoporose houve redução dos níveis de fosfatase alcalina óssea e da expressão da proteína anti-apoptótica Bcl-2 (p<0,05). Redução de DMO, densidade mineral cortical da diáfise distal, rigidez, resiliência e carga máxima da tíbia (p<0,05). Aumento da densidade radiográfica da epífise distal da tíbia e da expressão da proteína próapoptica Bax (p<0,05). No grupo alendronato houve redução da expressão de Bax (20 dias ) (p<0,05), aumento da expressão de Bcl-2 (30 dias) e da DMO da tíbia (20 e 30 dias) (p<0,05). EGb 1 e EGb2 (30 dias) aumentaram os níveis de fosfatase alcalina óssea (p<0,05). EGb2 aumentou a DMO (20 e 30 dias). EGb1 e EGb2 aumentaram a expressão de Bcl-2 (20 e 30 dias) (p<0.05). EGb2 (20 dias), EGb1 e EGb2 (30 dias) reduziram significativamente a expressão de Bax (p<0,05). EGb1 e EGb2 (20 e 30 dias) reduziram a densidade radiográfica (p<00,5). Entretanto os tratamentos com o alendronato de sódio e o EGb não alteraram de forma significativa os parâmetros biomecânicos. Conclusão: O EGb aumentou a DMO, a expressão de proteínas anti-apóptoticas e os níveis de fosfatase alcalina óssea. E ainda reduziu a densidade radiográfica e a expressão de proteínas pró-apoptóticas. Este estudo sugere que o EGb reduz a apoptose de células osteoblásticas e aumenta a formação de tecido ósseo, podendo ser um tratamento efetivo para a osteoporose induzida por glicocorticoides. / Introduction: Among the major mechanisms that lead to osteoporosis is the use of glucocorticoids. Glucocorticoids induce apoptosis in osteoblasts and reduce the total bone volume. Several products are used in the treatment of osteoporosis and among them, some natural products such as phytoestrogens. Studies with Ginkgo biloba extract (EGb) showed promising effects in the treatment of osteoporosis. This study aims to evaluate the effects of EGb on the apoptosis of osteoblasts, bone mineral density and bone structure of Wistar rats with glucocorticoid-induced osteoporosis. Methods: 60 rats were divided into 5 groups: control, osteoporosis, alendronate (alendronate sodium - 0.2 mg/kg/day), EGb1 (EGb 28mg/kg/day) and EGb2 (EGb 56mg/kg/day). Osteoporosis was induced by injections of dexamethasone. The bone alkaline phosphatase was measured in serum and immunohistochemical analysis of the proteins Bcl-2 and Bax were evaluated in histological sections of the mandible and femur. In the tibia were evaluated the bone mineral density (BMD), the radiographic density, the cortical mineral density and the biomechanical parameters. The control group was compared to the osteoporosis group ("t" test/ Mann Whitney). The other groups, except the controls, were analyzed by ANOVA followed by Tukey or Dunnett's T3 post hoc test (p <0.05). Results: In the osteoporosis group the levels of bone alkaline phosphatase and the anti-apoptotic protein Bcl-2 expression were significantly reduced. The BMD, cortical mineral density of the distal diaphysis, stiffness, resilience and load of the tibia reduced (p<0.05). The radiographic density of the distal tibial epiphysis and the expression of the pro-apoptic protein Bax increased (p<0.05). Alendronate group decreased the expression of Bax (20 days), and increased the expression of the Bcl-2 (30 days) and the BMD of the tibia (20 and 30 days) (p<0.05). EGb1 and EGb2 (30 days) increased the levels of bone alkaline phosphatase (p<0.05). EGb2 increased BMD (20 and 30 days) (p<0.05). EGb1 and EGb2 increased the expression of Bcl-2 (20 and 30 days) (p<0.05). EGb2 (20 days), EGb1 and EGb2 (30 days) reduced the expression of Bax (p<0.05). EGb1 and EGb2 (20 and 30 days) reduced radiographic density (p<0.05). However treatment with alendronate sodium and EGb did not increase significantly the biomechanical parameters. Conclusion: EGb increased BMD, the expression of anti-apoptotic proteins and bone alkaline phosphatase. The EGb reduced the radiographic density and the expression of pro-apoptotic proteins. This study suggests that EGb reduces apoptosis of osteoblastic cells and increases the formation of bone tissue. It suggests that the EGb may be effective in the treatment of osteoporosis

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-04-03T13:20:01Z No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-04-03T18:58:54Z (GMT) No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) / Made available in DSpace on 2017-04-03T18:58:54Z (GMT). No. of bitstreams: 1 ledamariliafonsecalucinda.pdf: 1555881 bytes, checksum: 1cf24f935ed553401a8a63db1a26cd5c (MD5) Previous issue date: 2009-04-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Introdução: A osteoporose é caracterizada pela diminuição do volume ósseo total e entre as principais causas da doença estão a redução de estrogênio, durante a menopausa e o uso crônico de glicocorticóides. Diversos produtos são usados em seu tratamento e entre estes alguns produtos naturais tais como os fitoestrógenos. Estudos “in vitro” com o extrato de Ginkgo biloba (EGb) demonstraram efeito estrogênico e ação protetora sobre os osteoblastos. Portanto o EGb poderia ser importante no tratamento da osteoporose. Métodos:72 ratas foram distribuídas em 6 grupos: controle, osteoporose, controle positivo (alendronato de sódio - 0,2mg/Kg/dia), EGb1 (14 mg EGb/Kg/dia), EGb2 (28 mg EGb/Kg/dia ) e EGb3 (56 mg EGb/Kg/dia). A osteoporose foi induzida através de injeções intramusculares de dexametasona na dose de 7mg/Kg, uma vez por semana, durante cinco semanas. Após a indução se iniciou o tratamento de 20 e 30 dias, exceto no grupo controle. Após a eutanásia, foram removidas as mandíbulas esquerdas para análise radiográfica digital, seguiu-se posteriormente a avaliação da cortical mandibular e do suporte ósseo periodontal (SOP) na superfície mesial e distal do primeiro molar mandibular. Para a análise histomorfométrica foram removidos as mandíbulas direitas e os fêmures direitos, para avaliação do percentual ósseo trabecular do fêmur (POT) e do percentual ósseo alveolar (POA) do segundo molar mandibular. Os grupos controles foram comparados aos grupos osteoporose (teste “t” Student). Os demais grupos, exceto os controles, foram analisados através do teste de ANOVA seguido do teste post hoc de Tukey ou Dunnett (p<0,05). Resultados: Foi observado através da análise radiográfica mandibular que o glicocorticóide causou diminuição do SOP, o alendronato de sódio apresentou recuperação do percentual do SOP, da mesma forma que o EGb nos tratamentos de 20 e 30 dias nas doses de 28mg/kg/dia e 56mg/kg/dia. O grupo EGb3 apresentou maior espessura cortical mandibular nos tratamentos de 20 e 30 dias. Através da histomorfometria dos animais tratados por 20 e 30 dias foi observado que o glicocorticóide causou diminuição no POT da epífise do fêmur e do POA mandibular (p,0,05). O grupo controle positivo apresentou aumento significativo do POT do fêmur (30 dias) quando comparado ao grupo osteoporose. O POA mandibular do grupo controle positivo apresentou aumento não significativo em relação ao grupo osteoporose (20 e 30 dias). Os grupos EGb 1 e EGb 2 apresentaram um aumento (p<0.05) do POT do fêmur quando comparados aos animais do grupo osteoporose (20 e 30dias). Da mesma forma os grupos EGb 2 e EGb 3 (20 dias) e os grupos EGb1, EGb2 e EGb3 (30 dias) apresentaram aumento significativo do POA mandibular quando comparado ao grupo osteoporose. Conclusão: O EGb recuperou a osteoporose induzida por glicocorticóide nos parâmetros avaliados neste estudo, sugerindo que o EGb possa ser efetivo no tratamento da osteoporose. / Introduction: Osteoporosis is a disease characterized by the reduction of bone volume. Among the main causes of osteoporosis is the reduction of the levels of estrogen and the chronic use of glucocorticoids. Several products are used on the osteoporosis treatment, including natural products, among them some promising alternatives as the phytoestrogens. In vitro studies showed that the extract of Ginkgo biloba (EGb) has a estrogenic effect and a protect action on the osteoblasts cell. Therefore the EGb may be important in the treatment of osteoporosis. Methods: 72 female rats were divided into six groups: control, osteoporosis, positive control (sodium alendronate-0.2mg/Kg/day), EGb1 (14mg/Kg/day), EGb2 (28mg/Kg/day) and (56mg/Kg/day). Osteoporosis induction was done trough intramuscular injection of dexamethasone in a dose of 7mg/Kg, once a week, during five weeks. Treatments were conducted after osteoporosis induction for 20 and 30 days. Following euthanazia, the left mandibles were removed for the digital radiographic analysis. The cortical and the periodontal bone support (PBS) at the mesial and distal surfaces of the first molar were analyzed. For the histomorphometric analysis the right femurs and mandibles were removed to evaluate the percentage of the trabecular bone (PTB) of the femur epiphysis and the percentage of the alveolar bone (PAB) of the interradicular septum of the second molar. The control groups were compared with the osteoporosis group (Student’s t-test). The others groups, except the control groups, were analyzed by ANOVA test followed by Tukey or Dunnett’s post-hoc test (p<0.05). Results: It was observed trough the mandibular radiographic analysis that the glucocorticoid caused the PBS reduction, the sodium alendronate recovered the percentage of PBS in the same way that EGb in the 28mg/kg/day and 56mg/kg/day doses in the treatments of 20 and 30 days. The EGb3 showed increase in the cortical mandibular thickness in the treatments of 20 and 30 days. Trough the histomorphometric analysis of the animals treated for 30 and 20 days, it was observed that glucocorticoids reduced the PTB of the femur and the PAB of the mandible (p<0,05). The positive control group (30 days) showed a statistical significant increase of the PTB of the femur when compared to the osteoporosis group (30 days). The PAB of the mandible in the positive control group showed no significant increase when compared to the osteoporosis group (20 and 30 days). The groups EGb1 and EGb2 groups showed an increase of the PTB of the femur when compared to the osteoporosis group (20 and 30 days), in the same way the EGb2 and EGb3 groups (20 days) and the EGb1, EGb2, and EGB3 groups (30 days) showed a statically significant increase of the PAB when compared to the osteoporosis group. Conclusion: The EGb recovered the effects oft glucocorticoid induced-osteoporosis in the evaluated parameters of this study, it suggests that the extract of Ginkgo biloba may be effective in the treatment of osteoporosis.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The goal of this project was to define mechanisms responsible for Na+ transport in two hormonally-sensitive epithelium, the bovine mammary gland and porcine vas deferens. Glucocorticoid stimulation in these epithelia results in a significant increase in amiloride-sensitive ion transport, suggesting regulation of the epithelial Na+ channel, ENaC. ENaC has typically been described as a heteromultimeric ion channel with at least three different types of subunits, the most common being , β, and γ. Glucocorticoid-induced regulation of these subunits at the transcriptional level appears to be very different in the porcine vas deferens as compared to the bovine mammary gland. The aims of the study in mammary epithelium were to elucidate the mechanisms by which apical electrolytes and cytokines compromise barrier function in mammary epithelium. The long term goal is to better understand and manage the interaction between ionic composition of milk and breakdown of the gland epithelium that occurs during mastitis. Our results suggest a causal link between changes in milk electrical conductivity and epithelial barrier breakdown that has not been appreciated previously. Results will provide benefits to dairy farmers by characterizing steps that might prevent the development of mastitis or hasten recovery. The aims of the study using porcine vas deferens epithelial cells include determining the time course, concentration- and structure-dependency for regulation of amiloride-sensitive ion flux by corticosteroids. Corticosteroids caused a concentration-dependent increase in amiloride-sensitive Isc with a rank order of potency of dexamethasone>prednisolone>cortisol. Hill analysis indicates steep concentration dependency. The corticosteroid-induced, amiloride-sensitive current is Na+ absorption as indicated by radiotracer flux measurements. Studies employing selective antagonists (spironolactone, mifepristone) define glucocorticoid receptor mediation. These results suggest that vas deferens epithelia are exquisitely sensitive to corticosteroid exposure. Observed changes in epithelial function in response to corticosteroid exposure would rapidly and chronically affect the luminal environment to which sperm are exposed. Thus, physiological and pharmacological corticosteroid exposure is expected to affect male fertility.

Endocrine Disrupting Chemicals (EDC) are exogenous agents that mimic endogenous hormone activity in the body. EDC exposure during the critical period of neonatal development can potentially cause life-long neurological, behavioural and physiological disease. This thesis focuses on the EDC Bisphenol A (BPA), a synthetic xenoestrogen widely prevalent in everyday materials that has significant environmental relevance given its ubiquitous presence in humans around the world. The central research question of my thesis is: Does perinatal exposure to BPA affect rat pup development? A rodent model was selected to study the effects of BPA on the adrenal component of the hypothalamic-pituitary-adrenal axis (HPA axis) stress pathway, which has not been extensively studied. Rat dams were divided into five groups (vehicle control (VEH), positive control diethylstilbestrol (DES), BPA 5, BPA 50 and BPA 500 μg/kg bw/day) and dosed daily throughout gestation and for four days of lactation. Rat pups were sacrificed at two time-points at the beginning and the end of the stress hyporesponsive period (SHRP), at postnatal day (PND) 5 and PND 15. Changes in three morphometric parameters (bodyweight, crown-rump (CR) length and anogenital distance (AGD) were assessed based on the factors of Treatment and Sex. Adrenal gland glucocorticoid receptor (GR) and 18SrRNA expression was determined by qPCR in male pups at PND 5 and PND 15. At PND 5, compared to the VEH group, the BPA 50 pups were significantly heavier (ANOVA, Dunnett’s post-hoc) and the DES and BPA 50 pups had significantly longer CR lengths (ANOVA, Dunnetts’ post-hoc). At PND 15, xenoestrogen treatment significantly influenced CR length (ANOVA). At both time-points, males had significantly longer AGD than females, as physiologically expected (ANOVA). Adrenal gland GR expression in male pups was not significantly affected by treatment, but there was an effect of treatment in18SrRNA gene expression at PND 5 (Kruskal-Wallis). Using the Ct method to determine GR and 18SrRNA fold changes, we cautiously suggest that our experimental doses resulted in a non-monotonic dose response to BPA in the PND 5 animals and a monotonic dose response to BPA exposure in the PND 15 animals. This study highly values the importance of investigating the effects of environmentally relevant, low dose BPA exposure during the critical window of development, given the little that is known about potentially permanent alterations to the stress pathway due to exposure during this delicate period of development.

Orientadores: José Andrés Yunes, Nilson Ivo Tonin Zanchin / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T06:00:33Z (GMT). No. of bitstreams: 1 Silveira_AndreBortolini_D.pdf: 16883235 bytes, checksum: e0759c48520d471791a5272349e1a837 (MD5) Previous issue date: 2013 / Resumo: A via PI3K está frequentemente hiperativada em células primárias de leucemia linfoide aguda T (LLA-T) pediátrica, característica previamente associada à resistência a glucocorticoides. Pacientes cujas células leucêmicas apresentam mutações em PTEN, o principal regulador negativo de PI3K, podem apresentar maior risco de falha na terapia de indução e recaída. Neste estudo, uma assinatura baseada em expressão gênica foi utilizada para acessar o nível de ativação da via PI3K em amostras diagnósticas de LLA-T. Nós identificamos Myc como um importante integrador da atividade de sinalização por PI3K e observamos que maior atividade da via está associada à resistência a glucocorticoides e pior prognóstico. O inibidor de PI3K AS605240 mostrou atividade antileucêmica e forte sinergismo com glucocorticoides tanto in vitro como em um modelo xenográfico de LLA-T em camundongos NOD/SCID. Em contraste, a inibição de PI3K resultou em antagonismo com metotrexato e daunorrubicina, drogas que atuam preferencialmente em células em divisão. Esta interação antagonística, no entanto, pôde ser revertida pelo uso de um esquema temporal específico de administração das drogas. Nossos dados indicam os potenciais benefícios e limitações para a incorporação de inibidores de PI3K na terapia da LLA-T / Abstract: The PI3K pathway is frequently hyperactivated in primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Activation of the PI3K pathway has been suggested as one mechanism of glucocorticoid resistance in T-ALL, and patients harboring mutations in the PI3K negative regulator PTEN may be at increased risk of induction failure and relapse. In this study, a PI3K gene expression signature was used as readout of PI3K activity in diagnostic T-ALL samples. We identified Myc as an important downstream integrator of PI3K pathway activity in T-ALL and found that higher PI3K activity is associated with glucocorticoid resistance and worse clinical outcome. The PI3K inhibitor AS605240 showed anti-leukemic activity and strong synergism with glucocorticoids both in vitro and in a NOD/SCID xenograft model of T-ALL. In contrast, PI3K inhibition showed antagonism with methotrexate and daunorubicin, drugs that preferentially target dividing cells. This antagonistic interaction, however, could be circumvented by the use of correct drug scheduling schemes. Our data indicate the potential benefits and difficulties for the incorporation of PI3K inhibitors in T-ALL therapy. OBSERVAÇÃOArquivo pdf com capa, página de rosto, folha de assinatura da banca examinadora, resumo e abstract foi editado segundo informação CCPG/002/2013 / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Resistance to glucocorticoid (GC) therapy results in poor prognosis for acute lymphoblastic leukemia (ALL) patients. Utilizing a whole genome shRNA screen our lab identified several novel mechanisms of GC resistance. My thesis work established that an orphan nuclear receptor, the Estrogen Related Receptor Beta (ESRRB), is critical for induction of apoptotic genes following treatment with the GC dexamethasone. ESRRB has mostly been implicated in maintenance of pluripotency in mouse embryonic stem cells. We find that repression of ESRRB results in GC resistance in ALL and define ESRRB as a novel cooperating transcription factor in GC-induced gene expression. We also show that agonists to ESRRB synergize with dexamethasone and increase dexamethasone induced apoptosis in relapse ALL patient samples. Interestingly, our shRNA screen identified another factor important in stem cell maintenance: SOX2. While we originally hypothesized that ESRRB and SOX2 may cooperate in ALL, RNA-sequencing studies revealed that these factors mediate GC resistance by independent mechanisms. Our data define SOX2 as a repressor of key signaling pathways in ALL. Upon SOX2 knockdown, we observe activation of pro-survival gene expression including activation of the MAPK pathway, which has previously been implicated in GC resistance. MAPK activation may be explained by an increase in EGFR expression observed in Sox2 knockdown cells and GC resistant patients, suggesting EGFR inhibitors may re-sensitize patients to GCs. Overall my thesis work identifies mechanisms of GC resistance in ALL and utilizes these findings to define novel therapeutic strategies for GC resistant ALL patients.

Zusammenfassung Katharina Regina Winkler Hämatologisch-immunologische Verlaufsuntersuchungen bei Kühen mit Gebärparese Medizinische Tierklinik der Veterinärmedizinischen Fakultät der Universität Leipzig Eingereicht im September 2014 100 Seiten, 41 Abbildungen, 13 Tabellen, 282 Literaturangaben, 17 Seiten Anhang Schlüsselwörter: Gebärparese, Stoffwechsel, Glucocorticoide, Endotoxin, Blutbild Problemstellung: Der Mineralstoffwechsel unterliegt immunologischen Einflüssen. Vitamin-D3 ist essentiell für die Antigen- und Zytokinsynthesen in den Blutzellen. Die Osteoblastenreifung wird durch Zytokine beeinflusst. Das tangiert die Gebärparese (GP) und erschließt potentiell prophylaktische sowie therapeutische Ansätze. Zielstellung: Es wurde geprüft, ob a) es Mineralstoffunterschiede bei der GP-Diagnose und im Verlauf zu früheren Studien gibt, b) die Parameter Endotoxin (ET), anti-Lipid A-IgG-Titer (ALA-IgG-Titer) und Haptoglobin (Hp) sowie Leuko- und Erythrogramme gesicherte Beziehungen zur GP sowie zu Mineralstoffen und immunologischen Parametern haben, c) die Therapie bei GP durch Glucocorticoide verbessert werden kann und d) Jungkühe mehr Geburtsstress und Belastungen des Ca-Pi-K-Stoffwechsels haben. Versuchsanordnung: Untersucht wurden 111 HF-Kühe bzw. Jungkühe: 21 GP-Kühe mit Grundbehandlung, 22 GP-Kühe mit zusätzlicher Dexamethason-21-iso-ni-cotinat-Therapie (Dexa-IN), 40 gesunde Kontrollkühe (KG) und 28 gesunde Jungkühe. Laborkontrollen erfolgten bei den GP-Kühen vor der Therapie, bei den KG 1 - 3 Tage post partum (d p. p.) sowie 1 d und 14 d nach dem Therapiedatum. Analysiert wurden neben Stoffwechselparametern Endotoxin (LAL-Test), ALA-IgG-Titer, Haptoglobin sowie das Leuko- und Erythrogramm. Ergebnisse: Die Erstbehandlung war bei 47 % ohne und 67 % mit Dexa-IN-Therapie erfolgreich; die Heilungsrate betrug 74 bzw. 82 %, d. h., die Dexa-IN-Therapie verbesserte das Behandlungsergebnis bei GP ohne Nebenwirkungen. 69,8 % der GP-Kühe hatten eine kombinierte Hypokalz- und Hypophosphatämie. 24 Stunden nach Beginn der Therapie waren beide Mineralstoffe in den GP-Gruppen wieder physiologisch. 11,6 % der GP-Kühe und 10,7 % der Jungkühe hatten eine Hypophosphatämie. Das ist offensichtlich eine Folge des Kalbestresses in diesen Gruppen. Die Mg-, Na- und Cl-Konzentrationen waren in allen Gruppen physiologisch. Mg korrelierte negativ mit Ca und Pi (p<0,01). Die K-Konzentrationen waren in den GP-Gruppen einen d p. p. signifikant niedriger als in den KG. Sie korrelierten mit Ca- und Pi in den GP-Gruppen mit 0,42 bis 0,48 (p<0,01). Auf stärkere Stresseinflüsse auf K wiesen Korrelationen zu Glucose, Bilirubin, eosinophile und basophile Granulozyten sowie Lymphozyten hin. Die Fe-Konzentrationen der GP- und KG-Kühe waren physiologisch. Fe korrelierte mit ALA-IgG-Titer gesichert negativ. Die ET-Konzentrationen ließen nur schwache Beziehungen zur GP erkennen, wie rET:Ca=-0,17 (p<0,05). ET korrelierte mit den ALA-IgG-Titern gesichert positiv (Dexa-IN-Gruppe). Die ALA-IgG-Titer differierten bei den Kühen nicht gesichert, sie korrelierten nicht mit Ca, aber mit Pi und mit der Mehrzahl der klinisch-chemischen und hämatologischen Parameter. Das zeigt die Entzündungseinflüsse auf den Pi-Stoffwechsel mit der Förderung von Hypophosphatämien. Die Hp-Konzentrationen streuten stark und waren in allen Gruppen am Diagnose- und noch mehr am Folgetag erhöht (p>0,05). Bei Jungkühen wies der höhere Anstieg auf stärkeren Kalbestress hin. Die Leukozytenzahl war am GP-Diagnosetag erhöht (Leukozytose; p>0,05) und sank zum Folgetag in den Normbereich ab. In der Dexa-IN-Gruppe war der Abfall am stärksten (p<0,05). Die Leukozytenzahl korrelierte gesichert negativ mit den ALA-IgG-Titer sowie in den GP-Gruppen mit den Pi- sowie Ca-Konzentrationen, ebenso die neutrophilen Granulozyten. Eosinophile, basophile Granulozyten sowie Lymphozyten sanken p. p. ab (p<0,05) und korrelierten gesichert mit Ca und Pi. Die GP-Kühe hatten am Diagnosetag eine Monozytose (p<0,05). Die Monozyten korrelierten mit den ALA-IgG-Titern, mit dem Pi und dem Ca gesichert negativ. Sie hatten die engsten Beziehungen zum Entzündungsgeschehen sowie den Pi- und Ca-Konzentrationen. Die CK-Aktivitäten waren am Diagnose- und am Folgetag gegenüber der KG signifikant erhöht. Mit Ca korrelierte die CK in allen Kuh-Gruppen gesichert negativ, mit Pi nur in der GP-Gruppe ohne Dexa-IN. Die CK stand in enger gesicherter Beziehung zu Entzündungsindikatoren. Hämoglobin war in den GP-Gruppen am Diagnosetag signifikant, der Hämatokrit und die Erythrozytenzahlen tendenziell gesteigert. Schlussfolgerungen: Die Studie zeigt bei GP-Kühen vielfältige Beziehungen des Ca-Pi-K-Stoffwechsels zu Entzündungsindikatoren und legt ursächliche Einflüsse nahe, besonders zu ALA-IgG-Titern und Monozyten. Sie können Ursachen für Hypophosphat- und Hypokalämie sein. Zusätzliche Dexa-IN-Therapie verbessert das Behandlungsergebnis.