Submitted by Tatiana Oliveira (tsilva@icict.fiocruz.br) on 2012-05-31T16:59:24Z No. of bitstreams: 1 bernardo_as_pereira_ioc_bp_002_2010.pdf: 3428909 bytes, checksum: 68f2b1087a42200dd708a3f10aac479e (MD5) / Made available in DSpace on 2012-05-31T16:59:24Z (GMT). No. of bitstreams: 1 bernardo_as_pereira_ioc_bp_002_2010.pdf: 3428909 bytes, checksum: 68f2b1087a42200dd708a3f10aac479e (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz.Instituto Oswaldo Cruz. Rio de janeiro, RJ, Brasil / A leishmaniose é uma doença antropozoonose que afeta 88 países, o que denota a importância da realização de estudos que permitem o desenvolvimento de novas estratégias de vacinação ou quimioterapias. Os agentes etiológicos dessa doença são espécies do gênero Leishmania, sendo que, no Brasil, a Leishmania (Leishimania) amazonensis está relacionada à forma tegumentar da leishmaniose e está expandindo sua área de distribuição geográfica. O modelo murino de infecção experimental tem sido largamente empregado nos estudos de leishmanioses, por permitir o controle das características do hospedeiro e a análise de aspectos específicos da doença. O presente estudo tem por objetivo verificar a atuação de fatores de interação parasito-hospedeiro nesse modelo de infecção utilizando linhagens de camundongos comdiferentes graus de susceptibilidade. Para tanto, efetuamos o seqüenciamento da extensão COOH-terminal de um tipo específico de cisteína proteinase (CP) do parasito, a CPB, e, em seguida, o mapeamento in silico de epitopos de MHC classe I nessa seqüência. Os epitopos preditos foram então sintetizados e utilizados em ensaios in vivo (vacinação) e in vitro (indução de blastogênese e de expressão de citocinas e efeitos sobre os linfócitos T CD4+ e CD8+). Alguns desses epitopos preditos demonstraram efeitos antigênicos nos ensaios in vitro, porém sem efeitos perceptíveis nos ensaios in vivo. Os epitopos preditos P4 e P5 induziram a blastogênese em culturas de células de camundongos BALB/c (mais susceptíveis a Leishmania), enquanto P2, P8 e P9 o fizeram em células de células de camundongos CBA (menos suscetíveis). Os epitopos P5, P6 e P8 também promoveram alterações nas porcentagens dos linfócitos CD4+ ou CD8+ em culturas de células de camundongos BALB/c. Quanto à indução da expressão de citocinas, os epitopos P1 e P2 (em BALB/c) e P2 e P3 (em CBA) induziram à expressão de citosinas relacionadas à resposta imune tipo Th1; P6 (em BALB/c) e P8 (em CBA) à expressão de citosinas da resposta Th2; e P4 (em BALB/c) e P9 (em CBA) à expressão de citosinas dos dois tipos de resposta imune. Ensaios de Molecular Doking foram utilizados para auxiliar na compreensão dos fenômenos de interação nos complexos epitoppos/MHC, apontando para padrões de interação associados aos padrões de indução da expressão de citocinas. Adicionalmente, foram efetuados ensaios de PCR em tempo real para se analisar os padrões de expressão de genes de moléculas de MHC do hospedeiro e de CPs do parasito ao longo da infecção, que indicam distinções na expressão de genes de MHC classes I e II entre as linhagens murinas e na expressão de CPs pelo parasito, o que pode estar relacionado às diferentes formas de progressão da infecção nessas linhagens. Os resultados obtidos nesse estudo complementam os dados da literatura sobre as interações parasito-hospedeiro na infecção experimental murina e apontam para novas estratégias de análise dessas interações em leishmaniose / Leishmaniasis is a disease that affects anthropozoonosis 88 countries, demonstrating the importance of studies that allow the development of new strategies for vaccination or chemotherapy. The etiologic agents of this disease are species of the genus Leishmania, and in Brazil, Leishmania (Leishimania) amazonensis is related to the cutaneous form of leishmaniasis and is expanding its geographical distribution. The murine model of experimental infection has been widely used in studies of leishmaniasis by allowing the control of the characteristics of the host and analysis of specific aspects of the disease. The present study aims to verify the performance factors of the host-parasite interaction in this infection model using mice strains comdiferentes degrees of susceptibility. Therefore, we performed by sequencing the COOH-terminal extension of a particular type of cysteine ​​proteinase (CP) of the parasite, CPB, and then, the in silico mapping of epitopes on MHC class I that sequence. The predicted epitopes were then synthesized and used in vivo assays (vaccination) and in vitro (induced blastogenesis and expression of cytokines and effects on T lymphocytes CD4 + and CD8 +). Some of these antigenic epitopes predicted effects demonstrated in vitro assays, but without noticeable effects in vivo assays .. The predicted epitopes P4 and P5 induced blastogenesis in cultured cells of BALB / c (most likely Leishmania), while P2, P8 and P9 cells did cell CBA mice (less likely). The epitopes P5, P6 and P8 also induced variations in the percentages of CD4 + or CD8 + cell cultures of BALB / c mice. The induction of expression of cytokines, the epitopes P1 and P2 (in BALB / c) and P3 and P2 (in CBA) to induce expression of cytokines related to Th1 type immune response, P6 (in BALB / c) and P8 (in CBA) to the expression of Th2 cytokines, and P4 (in BALB / c) and P9 (in CBA) to the expression of cytokines of both types of immune response. Molecular tests Doking were used to assist in understanding the complex interaction phenomena in epitoppos / MHC, pointing to patterns of interaction patterns associated with induction of cytokine expression. Additionally, PCR assays were performed in real time to examine the expression patterns of genes of the host MHC molecules and CPs along the parasite infection, indicating distinctions in the gene expression of MHC class I and II between the strains and expression of murine CPs the parasite, which can be related to different forms of progression of the infection in these strains. The results of this study complement the literature on host-parasite interactions in murine experimental infection and point to new strategies for analysis of these interactions in leishmaniasis

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on January 5, 2010). Vita. Thesis advisor: Habib Zaghouani. Includes bibliographical references.

Epidemiological evidence linking dietary folate deficiency and risk for colorectal cancer is conflicting. Studies using animal models indicate that timing, dose and presence of pre-malignant lesions will influence whether folate deficiency prevents or promotes tumor formation. In this thesis a new model of spontaneous tumor formation due to long-term dietary folate deficiency alone, in non-transgenic mice and without carcinogen induction, is developed. The mechanisms by which folate deficiency might influence cancer risk are also examined. / BALB/c mice, with or without a null allele in a key folate-metabolizing enzyme, Methylenetetrahydrofolate reductase (Mthfr ), develop intestinal tumors due to dietary folate deficiency alone. On folate-deficient (FD) diets, 12.5% of Mthfr+/+ mice and 28.1% of Mthfr+/- mice developed tumors; mice on control diet (CD) did not. C57B1/6 mice (a strain resistant to other methods of tumor induction) placed on the same diets for the same amount of time did not develop any tumors. To investigate possible mechanisms the levels of DNA damage (dUTP/dTTP ratio and p-H2AX staining) and DNA methylation (thin layer chromatography) were examined. FD BALB/c, but not C57B1/6 mice, had a trend towards increased dUTP/dTTP and DNA double-strand breaks and decreased global DNA methylation compared to CD mice. To determine why the FD diet affects the BALB/c and not the C57Bl/6 strain, the expression of genes involved in folate metabolism was examined. Several changes in gene expression were observed. In particular, BALB/c mice had increased Mthfr expression and MTHFR activity compared to C57Bl/6 mice. Increased MTHFR activity may deplete 5,10-methylenetetrahydrofolate supplies for the dTMP synthesis, increasing the dUMP levels and, possibly, DNA damage. The levels of several DNA repair genes were also examined. Two genes involved in base excision repair, Thymine DNA glycosylase (Tdg) and Apurinic/apyrimidinic endonuclease 1 (Apex1), were increased in FD C57B1/6 compared to FD BALB/c mice suggesting increased DNA repair capacity. / These results support the evidence that dietary folate deficiency promotes intestinal tumor formation possibly through increased DNA damage, with subsequent defects in DNA repair.

Development of specific DNA methylation patterns is required for normal spermatogenesis. DNA methyltransferases (DNMTs) use S-adenosylmethionine (SAM) produced in a pathway requiring 5,10-methylenetetrahydrofolate reductase (MTHFR). This thesis describes: testicular phenotype differences derived from Mthfr-deficiency in different mouse strains; the cellular Mthfr expression pattern during male germ cell development; and finally, changes to the DNA methylation of Mthfr-deficient sperm. Mthfr-deficient BALB/c, but not C57BL/6, mice have reduced neonatal germ cell proliferation but both have abnormal germ cells as adults. Germ cell MTHFR expression differed developmentally in parallel with DNMTs associated with de novo methylation. Sperm from mice with reduced Mthfr levels or dietary folate deficiency had differential DNA methylation at multiple loci, compared to wildtype mice, indicating that maintenance as well as acquisition of methylation can be altered by SAM-reduction. These results highlight the important role of folate in sperm development throughout life.

Spinal cord injury (SCI) results in a well-orchestrated inflammatory response which causes secondary tissue damage. Activated macrophages contribute to this cytotoxic response, which includes damage to neurons, glia and myelin, and tissue loss that worsens functional outcomes after SCI. However, activated macrophages in the spinal cord under other conditions are not cytotoxic, such as after intraspinal injection of lysophosphatidylcholine (LPC), a potent demyelinating agent. Recovery from SCI may be optimized by reducing the detrimental effects of macrophages while promoting their beneficial ones. Therefore, I compared spinal cord tissue, as well as purified macrophages, from mice after SCI (cytotoxic response) and intraspinal LPC injection (non-cytotoxic response). As a first step to carry out this work, I characterized the injury parameters for SCI contusion injury (i.e. injury force and spinal cord displacement) in mice using the Infinite Horizons impactor (Chapter 2). This lesioning model was used in other work for the thesis. The role T cells may play in mediating macrophage activation after LPC microinjection and SCI was also assessed using Nude mice (Chapter 3). Next, Affymetrix GeneChip analysis was carried out on spinal cord tissue obtained at the peak of the macrophage response after SCI and intraspinal LPC injection to identify potential candidate genes that may control the divergent inflammatory responses (Chapter 4). Several potential genes were identified. I next characterized the expression and role of one of these genes, MAPK activated protein kinase 2 (MK2), and showed that it mediates secondary tissue damage after SCI via several mechanisms (Chapter 5). The differences in gene expression profiles of macrophages purified from the spinal cord after SCI and LPC-injection were also assessed (Chapter 6). This microarray analysis of macrophages led to the identification of 10 novel candidate genes, two of which were validated at the protein level. Finally, I also examined the expression and role of secretory leukocyte protease inhibitor (SLPI) in SCI (Chapter 7). Using a combination of knockout/overexpressing transgenic mice and recombinant SLPI, I found that SLPI mediates protective anti-inflammatory effects after SCI. In conclusion, work done for this thesis has led to the identification of several novel molecules that influence the inflammatory response after injury and thus have led to the identification of potentially novel targets for the development of pharmacological approaches to treat acute SCI.

Dissertation (Ph.D.)--University of South Florida, 2006. / Includes vita. Includes bibliographical references (leaves 86-105). Also available online.

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A paracoccidioidomicose (PCM) é uma doença sistêmica de caráter granulomatoso, causada pelo fungo termodimórfico Paracoccidioides spp. A PCM é endêmica na America Latina e aproximadamente 80% do pacientes vivem no território brasileiro. O tratamento medicamentoso é eficiente, entretanto, é longo e vários pacientes acabam abandonando e recidivas são comuns neste grupo. A utilização de uma vacina terapêutica poderia resultar na redução do tempo de tratamento assim como, recuperar a resposta imune do hospedeiro frente ao fungo. As vacinas de DNA são uma abordagem promissora na imunoterapia e podem ser injetadas por via intramuscular, intradérmica ou via mucosa. As proteínas de choque térmico (HSPs) são proteínas que estão ligadas a homeostase celular e também possuem efeitos imunológicos em diversos casos como doenças infecciosas e autoimunes. No presente trabalho, analisamos o esquema vacinal terapêutico em camundongos BALB/c previamente infectados intratraquealmente com 3x105 leveduras de P. brasiliensis Pb18, 60 dias depois, submetidos a imunização com pcDNA3 contendo sequências codificadoras de PbHSP60 e/ou IL-12 murina e/ou vetor vazio. Foi observada redução significativa no número de unidades formadoras de colônia (UFCs) nos pulmões de camundongos imunizados com PbHSP60. Os grupos que receberam PbHSP60+pcDNA3 vazio ou PbHSP60x2 apresentaram os maiores índices de redução da cargas fúngicas. A inclusão do plasmídeo contendo o inserto de mIL-12, resultou em um efeito deletério. A análise dos cortes histológicos indicou que os animais vacinados apresentavam áreas bem preservadas e com poucos ou nenhum foco de granuloma. Detectamos um perfil de citocinas típico Th1/Th2. Nossos resultados sugerem que a imunização utilizando plasmídeo contendo o inserto HSP60, tem grande potencial vacinal / The paracoccidioidomycosis (PCM) is a systemic granulomatous disease of character, caused by the thermally dimorphic fungus Paracoccidioides spp. The PCM is endemic in Latin America and approximately 80% of patients are living in Brazil. The medical treatment is effective, however, is long and many patients end up abandoning and relapses are common in this group.The use of a therapeutic vaccine could result in the reducing time of treatment as well as recover the host immune response against the fungus. DNA vaccines are a promising approach for immunotherapy and can be injected by intramuscular, intradermal, or mucosal route. The heat shock proteins (HSPs) are proteins that are linked to cellular homeostasis and also have immunological effects in many cases as infectious and autoimmune diseases. In the present study, we analyzed the therapeutic vaccine schedule in BALB/c mice previously infected intratracheally with 3x105 yeast of P. brasiliensis strain 18, and 60 days after, undergoing immunization with pcDNA3 containing coding sequences PbHSP60 and / or murine IL-12 and / or empty vector. Significant reduction was observed in the number of colony forming units (CFU) in the lungs of mice immunized with PbHSP60. The groups that received empty pcDNA3 and PbHSP60 or PbHSP60x2 have higher rates of reduced fungal loads. The inclusion of the plasmid containing the insert mIL-12 resulted in a deleterious effect. The analysis of histological sections indicated that vaccinated animals had wellpreserved, with few or no focus of granuloma areas. It was detected a profile typical Th1/Th2 cytokines. Our results suggest that immunization using plasmid containing the insert HSP60 vaccine has great potential