Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-09-28T14:27:47Z No. of bitstreams: 1 2011 - Erica Cristina Rocha Roier.pdf: 982721 bytes, checksum: 24c0654d64de8a5b21a0eea5376f828c (MD5) / Made available in DSpace on 2016-09-28T14:27:47Z (GMT). No. of bitstreams: 1 2011 - Erica Cristina Rocha Roier.pdf: 982721 bytes, checksum: 24c0654d64de8a5b21a0eea5376f828c (MD5) Previous issue date: 2011-02-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / This study aimed to evaluate the prevalence of antibodies against Neorickettsia risticii in horses in the microregion of Itagua? RJ, show possible associated factors and identify the agent by molecular techniques. The study was conducted in the districts of Serop?dica, Itagua? and Mangaratiba, Rio de Janeiro. The 350 blood samples were obtained by convenience from horse properties belonging to the region. Serologic testing was performed by Immunofluorescence Assay (IFAI) for N. risticii, being considered positive samples titer 1:50. To evaluate risk factors associated with the presence of antibodies to N. risticii an epidemiological questionnaire was applied to the owners or guardians of animals, which aimed especially farm characteristics and management of animals. Hematological and clinical evaluation was performed for all animals. Molecular analysis by a Real Time PCR was performed from leukocyte cover. The prevalence of antibodies anti-N.risticii in horses of the region of Itagua? was 26.3% (92/350). The frequencies of IgG anti-N.rristicii were 52.2% (48/92) for titers of 1:50, zero for titers of 1:100, 13% (12/92) for titles of 1:200, 28.3% (26/92) for evidence of 1:400 and 6.5% (6 / 92) for titles of 1:800. The age and quality level of the properties were associated (p <0.05) with horses seropositivity for N. risticii. DNA from N. risticii was not detectable in samples of this study. Also no significant changes in the clinical evaluation of animals was found in relation to seropositivity for the agent. Despite showing association (p <0.05) between the seropositivity of the animals and changes in white blood cell of negative animals, these changes were not outside reference limits for the species evaluated. The existence of horses seropositive for N. risticii indicates the presence of this agent in the microregion of Itagua?. / O presente estudo teve por objetivo avaliar a preval?ncia de anticorpos contra Neorickettsia risticii em equinos na microrregi?o de Itagua?-RJ, demonstrar os poss?veis fatores associados e identificar o agente atrav?s de t?cnicas moleculares. O estudo foi conduzido nos munic?pios de Serop?dica, Itagua? e Mangaratiba, estado do Rio de Janeiro. As 350 amostras de sangue foram obtidas por conveni?ncia, das propriedades de cria??o de equinos pertencentes ? regi?o. O teste sorol?gico foi realizado atrav?s da Rea??o de Imunofluoresc?ncia Indireta (RIFI) para N. risticii, sendo consideradas positivas amostras com t?tulo 1:50. Para avalia??o dos fatores de risco associados ? presen?a de anticorpos de N. risticii, aplicou-se um question?rio epidemiol?gico com os propriet?rios e/ou respons?veis dos animais, destacando caracter?sticas da propriedade e manejo dos animais. Realizou-se hemograma e avalia??o cl?nica de todos os animais. As an?lises moleculares atrav?s da t?cnica de Real Time PCR foram realizadas a partir de capa leucocit?ria. A preval?ncia de anticorpos anti-N. risticii em equinos da microrregi?o de Itagua? foi de 26,3% (92/350) pela RIFI. As frequ?ncias de anticorpos IgG anti-N. risticii foram 52,2% (48/92) para titula??es de 1:50, zero para titula??es de 1:100, 13% (12/92) para t?tulos de 1:200, 28,3% (26/92) para t?tulos de 1:400 e 6,5% (6/92) para t?tulos de 1:800. A idade e o n?vel de qualidade das propriedades apresentaram associa??o (p<0,05) com a soropositividade dos equinos para N. risticii. N?o foi detectado o DNA de N. risticii nas amostras do presente estudo. Tamb?m n?o foram evidenciadas altera??es significativas na avalia??o clinica dos animais, em rela??o ? soropositividade para o agente. Apesar de haver associa??o (p<0,05) entre a soropositividade dos animais e altera??es no leucograma dos animais negativos, estas altera??es n?o estiveram fora dos limites de refer?ncia para a esp?cie animal avaliada. A exist?ncia de equinos soropositivos para N. risticii indica a circula??o desse agente na microrregi?o de Itagua?

Glycodelin is a 162 amino acid dimeric, glycosylated, secretory protein of the lipocalin superfamily. Its classification as a lipocalin(carriers of small hydrophobic molecules) is based mainly on the presence of lipocalin signature motifs in its primary sequence and no ligand for this protein has been identified till date. Glycodelin has 40-55% sequence identity with β-lactoglobulin which is the second type member of the lipocalin superfamily (the first being retinol binding protein (RBP). Glycodelin is primarily a primate specific protein (though there have been isolated reports of mRNA in mice and rats) with many isoforms secreted by various tissues, predominantly of the reproductive tract. These isoforms, being the product of the same gene, are identical in primary sequence and differ only in their glycosylation due to differences in tissue origin; hence they may be better addressed as glycoforms of glycodelin. The main glycoforms of glycodelin reported till now are GdA, GdS, GdM, GdF and GdC. Each glycoform of the protein has a varied function, dictated or modulated largely by the complex glycans on its surface. GdA, the most well studied glycoform of glycodelin, is secreted by the endometrium under progesterone control and accumulates in the amniotic fluid (from where it is isolated.). GdA has been subclassifed as an immunocalin (immuno-modulatory lipocalins) due to the many immuno-modulatory functions pertaining to tissue differentiation, implantation and angiogenesis and most sifnificantly, modulation of immune responses at ehe feto-maternal interface. The fetus expresses paternal allo-antigens on its surface and would be regarded as foreign or non-self by the maternal immune system. Yet the fetus is not rejected, and is in fact protected from attack by the maternal immune system by a variety of tolerogenic mechanisms. GdA is the most abundant secretary glycoprotein of the primate uterine compartment during implantation and early pregnancy. It has been shown to have inhibitory effect on innate as well as adaptive and humoral immune responses. It inhibits the proliferation of T and B cells, Nk cytoxocity and suppresses monocyte chemotaxis. It also skews the cytokine profile from Th1 to Th2 and inhibits IL1 and IL2 secretion from mitogenically stimulated lymphocyte and mononuclear cell cultures. In our laboratory, we have demonstrated earlier that the inhibitory effect of GdA on T cell proliferation is due to apoptosis being induced. The apoptotic signaling induced by GdA was found to be caspase dependent and follows the intrinsic mitochondrial stress induced pathway of apoptosis. Having determined the effect of glycodelin A on T cells, we wanted to look at its effect on other cells playing a role in immune responses. We decided to look at its effect, if any, on the innate immune system. Chapter 1 of the thesis describes our studies on the effect of GdA on monocytes. We have looked at the effect of GdA on primary monocytes isolated from blood of healthy human volunteers and found that GdA induces apoptosis of primary monocytes and this appears to be mediated through a caspase independent pathway. The mitochondrial membrane potential of primary monocytes was lost upon GdA treatment therefore the mitochondria seem to be involved in the apoptotic cascade. As the yield of monocytes from peripheral blood is very low, further studies on the effect of GdA on monocytes were carried out using a human monocytic cell line, THP1, as a model system. We have demonstrated the GdA is able to inhibit the proliferation of these cells and also induce apoptosis in them. We also found that this signaling is partially caspase-dependent and involvement of other caspase independent pathways is possible. Further, we have shown that there is no effect of GdA on the phagocytic ability of these cells after differentiation into the macrophage lineage. However, when added before differentiation, glycodelin is able to inhibit the phagocytic ability of THP1 cells. We also found that THP1 cells were relatively resistant to GdA-induced apoptosis post differentiation into macrophages. We have also looked at the effect of GdA on B cells using primary B cells as well as a B cell line U266B1 as our model system. GdA was shown to inhibit the proliferation of primary B cells as well as of the cell line. The protein was not able to induce apoptosis in the primary cells (both activated as well as unactivated cells) as well as in the cell line. Treatment of the cells with MAP kinase inhibitors also did not render them susceptible to GdA induced apoptosis(as has been seen in the case of U937 cells). U266B1 cells remained relatively resistant to GdA-induced apoptosis even when treated for long periods. They did not undergo significant necrosis uponGdA treatment even though the proliferation of these cells was inhibited by the protein. We were surprised to find that there was loss of mitochondrial membrane potential of the cells upon GdA treatment even when there was no cell death. The reason for this is not clear. The inhibition of proliferation of B cells by GdA does not involve caspases and the signalilng induced by GdA in these cells seems to be different to that induced in T cells atleast downstream of the mitochondria as the cells cannot proliferate in presence of GdA but seem immune to further damage or apoptosis. These studies have been described in chapter 2 of the thesis. The third and final chapter of the thesis deals with our investigation into the effect of GdA on Nk cells. GdA, in an earlier report, has been shown to inhibit the activity of circulatory NK cells. However, the mechanism of this action has not been delineated. We made attempts to determine the effect of GdA on NK cells using a human NK cell line YT Indy as our model system, as isolation and culture of primary Nk cells in good numbers is difficult. Preliminary studies revealed that GdA triggered apoptosis in these cells. However, the process was found to be caspase independent. Another surprising finding was that GdA did not bring about significant loss of mitochondrial membrane potential of these cells, implying that the involvement of mitochondria in the apoptotic signaling in these cells may be at the later stages, as amplifiers rather than initiators, as has been seen in the case of T cells and monocytes.

Surfactant proteins, SP-A and SP-D, are collagen-containing calcium-dependent (C-type) lectins, called, collectins. Their primary structure has four regions: a cysteine-linked N- terminal region involved in multimerization, a collagen region composed of Gly-X-Y repeats, coiled-coil neck region, and the C-terminal carbohydrate recognition domains (CRD) or C-type lectin domain. SP-A looks like a bouquet, while SP-D is a cruciform- like structure, with four arms of equal length. SP-A and SP-D have been shown to act as innate immune molecules at pulmonary as well as extra-pulmonary sites by binding to pathogens, allergens and apoptotic/necrotic cells via their CRD region. SP-A and SP-D can induce pathogen neutralization and enhanced phagocytosis. In addition, SP-A and SP-D can interact via CRDs with allergens and dampen allergic reaction in vitro and in vivo. This thesis examines in vitro interaction of a recombinant fragment of human SP-D containing neck and CRD regions (rhSP-D) with IAV and Aspergillus fumigatus, in addition to characterizing a dichotomy of the effects of SP-A and SP-D on dendritic cells in an attempt to explain how SP-A and SP-D modulate DC functions differentially. Experiments involving interaction of rhSP-D with IAV pandemic strain show that it can be a restrictive factor against the virus, in addition to modulating immune response by a macrophage cell line. The rhSP-D can have anti-A. fumigatus effect directly and indirectly in the context of pathogen as well as allergen. A comparison has been made between two recombinant fragments of SP-D that have been expressed with and without 8 Gly-X-Y repeats for their fungistatic properties. The effects of SP-A and SP-D on cultured DC maturation, and effector cytokine and proliferative response of co-cultured cells have also been examined in vitro.

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L'apoptose lymphocytaire joue un rôle essentiel dans le contrôle de la réponse immunitaire et de la prolifération cellulaire. De nombreuses voies interviennent dans sa régulation, dont certaines dépendantes de l’oncogène RAS. Un défaut d'apoptose lymphocytaire induit l'apparition de maladies auto-immunes et lympho-prolifératives comme l'Autoimmune LymphoProliferative Syndrome (ALPS). L'ALPS fait suite à des anomalies du principal récepteur membranaire de mort FAS, pivot de l'apoptose lymphocytaire. Le RAS-Associated Lymphoproliferative Disease (RALD) est une entité décrite récemment, se rapprochant de l'ALPS par la symptomatologie et la physiopathologie sous-jacente. Cependant, dans le RALD, le défaut d'apoptose lymphocytaire n’est pas lié à des mutations de FAS mais à une hyperactivation de la voie RAS, mettant ainsi en lumière le rôle essentiel de cette voie dans la régulation du processus en question. Dans les Leucémies Myélo-Monocytaires Juvéniles Chroniques (JMML), les mêmes mutations que celles observées dans les RALD sont trouvées, sur les mêmes populations cellulaires. Il existe une hétérogénéité clinique et biologique au sein des JMML, certaines étant indolentes (LS-JMML) et d'autres sévères (S-JMML). A cette hétérogénéité au sein même des JMML s'ajoute celle observée entre JMML et RALD. L'objectif de ce travail a été de comprendre les tenants des différences phénotypiques observées, au travers du scope de l'apoptose des lymphocytes T activés, en comparant les trois entités résultant de mutations activatrices de RAS dans des cellules pluripotentes hématopoïétiques : RALD, LS-JMML et S-JMML. Nous rapportons des conséquences distinctes pour des mutations identiques ou équivalentes, avec différentes voies de l'apoptose touchées, différenciant les phénotypes induits. Ce travail a permis de démontrer que l’hyperactivation de la voie RAS seule n’entraîne pas nécessairement une dérégulation de la réponse immunitaire T. Des événements additionnels aux mutations présentes sont nécessaires au développement des symptômes. Ces événements ont bien des conséquences sur l'apoptose lymphocytaire, au niveau post-traductionnel, qu’ils concernent la voie RAS ou non. Les différences observées entre les trois phénotypes sur le plan expérimental pourraient être une aide au pronostic. De plus, ce travail ouvre la voie à l'identification en détails des facteurs additionnels et voies défaillantes et permettrait ainsi d'obtenir des thérapeutiques spécifiques actuellement inexistantes. / Lymphocytes apoptosis is essential in maintaining homeostasis and avoiding abnormal proliferation. When defective, autoimmune diseases as the Autoimmune LymphoProliferative Syndrome (ALPS), due to mutations of the death receptor FAS, can occur. Several pathways are important actors influencing the apoptosis cascade, including the RAS proto-oncogene signaling. The RAS Associated Lymphoproliferative Disease (RALD) is a newly described entity, similar to ALPS but with RAS mutations instead of FAS mutations, enlightening the primary role of RAS in apoptosis regulation. Interestingly, the same RAS mutations as observed in RALD are also the cause of a malignant proliferation, the Juvenile Myelo Monocytic Leukemia (JMML). In the case of JMML, RAS mutations can lead either to a mild (LS-JMML) or a severe (S-JMML) phenotype. Thus, three different phenotypes can be caused by the same oncogenic RAS mutations. In order to better understand and characterize the influence of oncogenic RAS mutations in lymphocytes’ apoptosis we studied it in patients presenting with RALD, LS-JMML and JMML. We showed that isolated RAS hyperactivity is not sufficient to induce an immune deregulation. Additional factors are required to do so. These factors influence both mitochondrial and extrinsic apoptosis pathways at a post-transcriptional level. They are due to probable genetic events, and their identification can lead to new therapeutic strategies. Furthermore, activated lymphocytes’ in vitro apoptosis assessment can help differentiating the three phenotypes and thus facilitate prognosis prediction.