[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Apoptosis is a morphologically distinct form of cell death that is involved in the regulation of normal and aberrant cell systems. The complexities of the apoptotic cell death pathway arise from variation in both the cellular specialisation and initial stimulus. The corpus luteum (CL) is an endocrine gland that whilst critical to the maintenance of pregnancy in the rat, regresses at the completion of each oestrous cycle and pregnancy. This regression is facilitated through apoptosis; though, the stimulus and factors involved in the apoptotic pathway are poorly understood. Previous studies suggest that CL regression is not initiated through failure of luteotrophic support, but rather the active production of a luteolytic factor, of which tumour necrosis factor -alpha (TNFα) is one possible candidate. Several publications have reported the participation of the immune system in ovarian events. There is evidence that TNFα expression within the ovary is coordinated between cells of the immune system and the hormonal regulation of the CL. This study has focussed on the role of TNFα in CL apoptosis and the factors involved in this apoptotic pathway. TNFα-induced cell death is governed by the presence of the two TNFα receptors (TNFR) and several second messenger systems that include; the sphingolipids, mitogen-activated protein (MAP) kinases, nitric oxide (NO), nuclear factor-kappaB (NF-κB) and the caspases. These factors and their interactions were assessed in the rat CL during pregnancy and post-partum, and in vitro. Apoptosis was measured through the analysis of DNA fragmentation using DNA 3’ end labelling and single cell electrophoresis (COMET assay). Assessment of mRNA and protein expression was through Real-time RT-PCR and Western blot analysis; proteins were localised within the CL by immunocytochemistry. In addition, specific measurement of sphingolipid expression and nitric oxide (NO) production was by high performance liquid chromatography (HPLC) and NO assay respectively. Following parturition, TNFα mRNA and protein expression increased corresponding to the onset of CL apoptosis and increased expression of the chemotactic factor monocyte chemoattractant protein -1 (MCP-1). Furthermore, CL apoptosis was induced by treatment with recombinant TNFα in a time- and dose-dependent manner. A similar effect was observed in isolated luteal cells. Simultaneously, the functional regression of the CL was assessed by measurement of both progesterone synthesis and steroidogenic acute regulatory (StAR) protein expression. StAR mRNA and protein expression declined toward parturition in vivo. Immunocytochemical studies revealed the presence of TNFα receptors 1 (TNFR1) and 2 (TNFR2) in luteal cells. Furthermore, TNFR mRNA was isolated from CL throughout pregnancy and post-partum. Subsequently, the role of the sphingolipids ceramide and sphingosine was examined during CL apoptosis in vitro. Ceramide and sphingosine were found to be potent apoptotic agents when administered in vitro (50µM). The downstream signal transduction of TNFα and ceramide was assessed through MAP kinase expression. Both TNFα and ceramide increased expression of the pro-apoptotic p38 MAP kinase with no change to the non-apoptotic extracellular signal-related kinase (ERK1&2). Despite previous reports of c-Jun NH2 terminal kinase (JNK) involvement in the cell death pathway, JNK expression was not evident in the rat CL. The caspases are a family of cysteine proteases central to the regulation and execution of apoptosis. General inhibition of the caspase cascade in vitro was effective in preventing apoptosis regardless of the apoptotic stimulus (TNFα, ceramide and sphingosine), suggesting that this pathway is central to CL apoptosis. Specific inhibition of several caspases produced a varying effect; inhibition of caspases 3, 6 and 8 significantly reduced the level of TNFα-induced apoptosis, thus supporting their classification as either regulatory or effector caspases. NO is endowed with the unique ability to initiate and to block apoptosis and this dichotomy extends to the cytotoxic actions of TNFα. Inhibition of NO production by treating CL with L-NAME prevented the onset of apoptosis, whilst NO production increased in response to increasing levels of apoptosis following trophic withdrawal. However, this effect was not seen during TNFα-induced apoptosis, suggesting that the actions of NO are independent of TNFα. The data presented within this study examine multiple elements of the TNFα cell death pathway in a single system. The results suggest that these elements are involved in TNFα signal transduction and furthermore, in rat CL apoptosis. It can be said that TNFα plays an active role in CL regression through the activation of the caspases, the sphingolipids and the MAP kinases.

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