Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references (leaves 179-187). Also available online.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, / Typescript. Bibliography: leaves 44-68.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 99-113.

Thesis (M.S.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 49 pages. Includes Vita. Includes bibliographical references.

Orientador: Aulus Cavalieri Carciofi / Banca: Helio Jose Montassier / Banca: Iracilda Zeppone Carlos / Resumo: Vários derivados da parede celular da levedura Saccharomyces cerevisiae conhecidamente agem sobre a imunidade, no entanto a ação, especialmente da fração beta-glucano, foi pouco demonstrada em cães. Para o estudo dos possíveis efeitos sobre a imunidade na espécie canina foram empregadas quatro dietas isonutrientes, contendo uma fonte de parede celular de levedura (PCL), duas fontes de beta-glucano (BG1 e BG2) e uma dieta controle (CT). Foram utilizados 24 cães da raça beagle, adultos, divididos em quatro grupos de seis animais. As dietas foram fornecidas por um período total de 126 dias e as avaliações incluíram hemograma e avaliações bioquímicas, dosagem de anticorpos anti-Leptospira, imunofenotipagem de linfócitos sanguíneos, avaliação da concentração de IgA em fezes, teste de hipersensibilidade cutânea tardia e dosagens de citocinas em sobrenadante de cultura celular. Os animais foram submetidos a desafio antigênico com vacina contra leptospirose no dia 42. Os dados foram avaliados pelo procedimento GLM do SAS, sendo as médias comparadas pelo teste de Tukey (p≤0,1). Nos exames bioquímicos houve discreta variação entre os tratamentos e ao longo dos dias. No hemograma notou-se aumento dos linfócitos para BG2. A dosagem de anticorpos anti-Leptospira, mostrou baixos títulos, não havendo boa resposta à vacinação. A imunofenotipagem revelou um aumento dos linfócitos T totais, T helper, T citotóxicos e linfócitos B no grupo BG2 e de linfócitos T citotóxicos e linfócitos B para o grupo PCL. Apesar da variação da concentração de IgA fecal ao longo dos dias, os tratamentos não influenciaram tais parâmetros. O teste de hipersensibilidade cutânea tardia mostrou um aumento na resposta à inoculação da vacina, para os grupos PCL e BG2. Na dosagem de citocinas em sobrenadante de cultura celular, apenas foi observada diferença na quantificação de TNF-α, ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Some products from the cell wall of the yeast Saccharomyces cerevisiae are known to act on the immunity, however this action, especially of the beta-glucan fraction, has never been demonstrated in dogs. To study these effects on the immunity of dogs four isonutrient diets were made, containing one source of yeast cell wall (YCW), two sources of beta-glucan (BG1 and BG2) and a control diet (CT). 24 adult beagle dogs were used, divided in four groups of six animals. Diets were given for a 126 days period. Evaluations included complete blood count and biochemistry profile, quantification of antibodies against Leptospira, immunophenotyping of blood lymphocytes, IgA concentration in feces, delayed-type hypersensitivity test and quantification of cytokines in cell culture supernatant. Animals were exposed to antigen challenge, by the vaccine against leptospirosis on day 42. Data were analyzed by the GLM procedure of the SAS software and the means were compared by the Tukey test (p<0,01). Biochemistry profile showed slight differences among the groups. A increase in lymphocyte count was observed for BG2 treatment. Quantification of antibodies against Leptospira showed low titles, with poor response to vaccination. Immunophenotyping revealed an increase during the time in total T cells, helper and cytotoxic T cells, and B lymphocytes for BG2 and of cytotoxic T cells and B lymphocytes for YCW group. Despite the variation in fecal IgA concentration during the time, treatments did not influence these parameters. Delayed-type hypersensitivity test showed an increased response to the vaccine inoculation, for YCW and BG2 groups. In the quantification of cytokines in cell culture supernatant the only difference observed was in TNF-α concentration, being BG2 higher than CT. We concluded that both yeast cell wall and beta-glucan fraction act on dogs' immunity / Mestre

Made available in DSpace on 2014-06-11T19:23:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-23Bitstream added on 2014-06-13T20:11:36Z : No. of bitstreams: 1 zaine_l_me_jabo.pdf: 606188 bytes, checksum: da0b30ee696bae955544945c1be515d9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Biorigin Sa / Vários derivados da parede celular da levedura Saccharomyces cerevisiae conhecidamente agem sobre a imunidade, no entanto a ação, especialmente da fração beta-glucano, foi pouco demonstrada em cães. Para o estudo dos possíveis efeitos sobre a imunidade na espécie canina foram empregadas quatro dietas isonutrientes, contendo uma fonte de parede celular de levedura (PCL), duas fontes de beta-glucano (BG1 e BG2) e uma dieta controle (CT). Foram utilizados 24 cães da raça beagle, adultos, divididos em quatro grupos de seis animais. As dietas foram fornecidas por um período total de 126 dias e as avaliações incluíram hemograma e avaliações bioquímicas, dosagem de anticorpos anti-Leptospira, imunofenotipagem de linfócitos sanguíneos, avaliação da concentração de IgA em fezes, teste de hipersensibilidade cutânea tardia e dosagens de citocinas em sobrenadante de cultura celular. Os animais foram submetidos a desafio antigênico com vacina contra leptospirose no dia 42. Os dados foram avaliados pelo procedimento GLM do SAS, sendo as médias comparadas pelo teste de Tukey (p≤0,1). Nos exames bioquímicos houve discreta variação entre os tratamentos e ao longo dos dias. No hemograma notou-se aumento dos linfócitos para BG2. A dosagem de anticorpos anti-Leptospira, mostrou baixos títulos, não havendo boa resposta à vacinação. A imunofenotipagem revelou um aumento dos linfócitos T totais, T helper, T citotóxicos e linfócitos B no grupo BG2 e de linfócitos T citotóxicos e linfócitos B para o grupo PCL. Apesar da variação da concentração de IgA fecal ao longo dos dias, os tratamentos não influenciaram tais parâmetros. O teste de hipersensibilidade cutânea tardia mostrou um aumento na resposta à inoculação da vacina, para os grupos PCL e BG2. Na dosagem de citocinas em sobrenadante de cultura celular, apenas foi observada diferença na quantificação de TNF-α,... / Some products from the cell wall of the yeast Saccharomyces cerevisiae are known to act on the immunity, however this action, especially of the beta-glucan fraction, has never been demonstrated in dogs. To study these effects on the immunity of dogs four isonutrient diets were made, containing one source of yeast cell wall (YCW), two sources of beta-glucan (BG1 and BG2) and a control diet (CT). 24 adult beagle dogs were used, divided in four groups of six animals. Diets were given for a 126 days period. Evaluations included complete blood count and biochemistry profile, quantification of antibodies against Leptospira, immunophenotyping of blood lymphocytes, IgA concentration in feces, delayed-type hypersensitivity test and quantification of cytokines in cell culture supernatant. Animals were exposed to antigen challenge, by the vaccine against leptospirosis on day 42. Data were analyzed by the GLM procedure of the SAS software and the means were compared by the Tukey test (p<0,01). Biochemistry profile showed slight differences among the groups. A increase in lymphocyte count was observed for BG2 treatment. Quantification of antibodies against Leptospira showed low titles, with poor response to vaccination. Immunophenotyping revealed an increase during the time in total T cells, helper and cytotoxic T cells, and B lymphocytes for BG2 and of cytotoxic T cells and B lymphocytes for YCW group. Despite the variation in fecal IgA concentration during the time, treatments did not influence these parameters. Delayed-type hypersensitivity test showed an increased response to the vaccine inoculation, for YCW and BG2 groups. In the quantification of cytokines in cell culture supernatant the only difference observed was in TNF-α concentration, being BG2 higher than CT. We concluded that both yeast cell wall and beta-glucan fraction act on dogs` immunity

Resumo: O autor avaliou a capacidade do hidróxido de cálcio em neutralizar o LPS de Pseudomonas aeruginosa, através de duas metodologias, a liberação de óxido nítrico e Fator de Necrose Tumoral Alfa (TNF-a) em cultura de macrófagos peritoneais de camundondos. O autor concluiu que o LPS bacteriano é um potente estimulador da produção de NO e TNF-a e que o tratamento deste LPS com hidróxido de cálcio causa sua inativação. / Abstract: It was evaluated the calcium hydroxide in neutalysing "Pseudomonas aeruginosa"'s LPS. Two methodologies were used; NO and TNF-a liberation in macrophage's rat culture. It was concluded that Ca(OH)2 inhibitted TNF-a and NO liberation. / Orientador: Renato de Toledo Leonardo / Coorientador: Iracilda Zeponi Carlos / Banca: Fábio Luiz Camargo Villela Berbert / Banca: Caio César Randi Ferraz / Banca: Odilon Mattos Rasquin / Doutor

Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas. Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T15:36:26Z (GMT). No. of bitstreams: 1 Romanatto_Talita_D.pdf: 1337280 bytes, checksum: 7ec7fd5e6f446c2f8f8a42019123e0d4 (MD5) Previous issue date: 2009 / Resumo: O aumento da prevalência de obesidade em várias regiões do planeta é um dos mais importantes fenômenos clínico-epidemiológicos da atualidade. Fatores como a mudança do hábito alimentar e o estilo de vida sedentário, aliados a determinantes genéticos ainda pouco conhecidos desempenham um papel relevante na patogênese dessa doença. Nos últimos quinze anos, desde o descobrimento do hormônio leptina, avanços consideráveis foram obtidos na caracterização dos mecanismos hipotalâmicos do controle da ingestão alimentar e da termogênese. Em um estudo recente demonstrou-se que o consumo de uma dieta rica, em gordura induz a expressão de várias citocinas pró-inflamatórias no hipotálamo e que a inibição da via de sinalização intracelular da serina quinase JNK é capaz de reverter parcialmente algumas das conseqüências clínicas do consumo dessa dieta (De Souza et al, 2005). No presente estudo avaliou-se a importância do receptor 1 de TNF-cc(TNFRI) na gênese do processo inflamatório desencadeado pela dieta rica em gordura. O TNFR1 é responsável pela maioria dos efeitos do TNF-ct, no entanto no contexto da obesidade induzida por dieta, a função desse receptor não está completamente esclarecida. Para tanto, camundongos knockout (KO) para o TNFR1 e seu respectivo background genético, C57BL6, foram tratados por 8 semanas com dieta hiperlipídica e observamos que o TNFR1 KO está protegido da obesidade induzida por dieta por meio de aumento na termogênese. Em ambas as dietas, padrão e hiperlipídica (HF), TNFR1 KO ganha menos peso apesar de aumento na ingestão alimentar. Adiposidade e diâmetro dos adipócitos estão reduzidos, assim como as concentrações sanguíneas de insulina e leptina. TNFR1 KO estão protegidos de resistência hipotalâmica à via da leptina por meio de preservação da sinalização da leptina através de JAK2, STAT3 e FOX01. TNFR1 KO apresentam aumento de termogênese, pelo aumento do consumo de 02, aumento da expressão de UCP-1 e UCP-3, no tecido adiposo marrom e músculo esquelético, respectivamente, e aumento do consumo de O2 de mitocôndrias isoladas de músculo. Isso demonstra que a via de sinalização do TNF-a pelo TNFR1 representa um importante mecanismo envolvido na termogênese deficiente associada à obesidade. / Abstract: In diet-induced obesity, hypothalamic and systemic inflammatory factors trigger intracellular mechanisms that lead to resistance to the main adipostatic hormones, leptin and insulin. Tumor necrosis factor-alpha (TNF-a) is one of the main inflammatory factors produced during this process and its mechanistic role as an inducer of leptin and insulin resistance has been widely investigated. Most of TNF-a inflammatory signals are delivered by TNFR1; however, the role played by this receptor in the context of obesity-associated inflammation is not completely known. Here, we show that TNFR1 knockout (TNFR1 KO) mice are protected from diet-induced obesity due to increased thermogenesis. Under standard rodent chow or ligh-fat diet, TNFR1 KO gain significantly less body mass in spite of increased caloric intake. Visceral adiposity and mean adipocyte diameter are reduced and Dlood concentrations of insulin and leptin are lower. Protection from hypothalamic eptin resistance is evidenced by increased leptin-induced suppression of food ntake and preserved activation of leptin signal transduction through JAK2, STAT3 jnd FOX01. Under high fat diet, TNFR1 KO mice present a significantly increased expression of the thermogenesis-related neurotransmitter, TRH. Further evidence if increased thermogenesis includes the increased 02 consumption and C02 traduction in respirometry measurements, increased expressions of UCP1 and JCP3 in brown adipose tissue and skeletal muscle, respectively, and increased 02 onsumption by isolated skeletal muscle fiber mitochondria. This demonstrates that 'NF-ct signaling through TNFR1 is an important mechanism involved in obesity-ssociated defective thermogenesis. / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica