Sporothrix shenckii, um fungo dimórfico, termo-dependente, é encontrado na natureza associado com matéria orgânica em decomposição, como, espinhos, folhas secas, madeira e também na água. Este fungo é o agente causador da esporotricose, que ocorre com maior freqüência em pessoas que trabalham com o solo e vegetais contaminados com os esporos do fungo, sendo considerada uma doença ocupacional. A contaminação acontece com a inoculação traumática do material contaminado e em alguns casos por inalação dos esporos. Quando infecta o organismo humano, bem como outros mamíferos ou quando cultivado a 37°C, passa para a forma de levedura. É comum em áreas de clima tropical e subtropical, com alta umidade. Apresenta distribuição mundial, sendo que, atualmente no Brasil, são relatados casos em São Paulo, Rio de Janeiro e no Rio Grande do Sul. No Rio de Janeiro, a esporotricose tem sido relatada como doença endêmica, onde o agente etiológico é transmitido de gatos para homens. A esporotricose apresenta caráter crônico, polimórfico e com formação granulamatosa. As formas clínicas e a patogênese dependem do sítio de inoculação do fungo e da resposta imunológica do hospedeiro. O mecanismo de defesa e suscetibilidade do organismo, bem como a disseminação da infecção por este fungo ainda não são totalmente conhecidos. O estado de saúde do paciente contribui diretamente para o estabelecimento da doença, sendo que, alcoolismo, diabete, imunidade comprometida e uso extensivo de drogas imunosupressoras são fatores que podem predispor à infecções severas. Neste trabalho estudamos in vitro a interação do S. schenckii com macrófagos murinos. Por meio de técnicas de fagocitose, ELISA, dosagem de óxido nítrico, viabilidade de leveduras, citometria de fluxo e RT-PCR foram realizados ensaios da atividade de macrófagos. Desta forma foram gerados alguns dados para o melhor entendimento da resposta de defesa nesta doença. Nos estudos realizados, concluímos que os anticorpos gerados nos camundongos pela infecção por S. schenckii foram capazes de induzir a fagocitose do fungo. Também, após a fagocitose das leveduras pelos macrófagos, incubados com laminarina ou manana, houve inibição significativa da produção de TNF-α, indicando que os receptores dectina-l e manose, respectivamente, estão relacionados à produção desta citocina. O soro, contendo os anticorpos do animal infectado, também induziu forte produção desta citocina, mostrando que a entrada do fungo via receptor Fc pode também induzir a produção dessa citocina. Verificamos ainda que a fagocitose de leveduras, pelos macrófagos, na presença de soro de camundongo infectado, induziu alta produção de IL-10. Quando analisamos a expressão de genes presentes nos macrófagos, observamos forte expressão de TLR-2 e MyD-88, na presença das leveduras. Já a expressão de iNOS pelos macrófagos, observamos que foi maior na presença do fungo somente quando as leveduras foram incubadas com o soro, após o período de 72 horas. Em seguida, ao dosar a produção de Óxido Nítrico (NO), verificamos no período de 3 dias de incubação, que houve redução significativa na concentração de NO produzida pelos macrófagos, quando estes foram incubados com o fungo. Porém, quando as leveduras foram opsonizadas pelo soro, houve aumento da produção de NO após 72 horas de incubação. Outra verificação importante foi que os macrófagos foram capazes de destruir as leveduras fagocitadas na presença de soro. / Sporothrix schenckii is a dimorfic fungus, term-dependent, find in nature associated with organic material at decomposition, like thorns, wheat leaves, wood and water. This is the fungi agent that causes sporothricosis, which happens with more frequency with people that work with land and green, contaminated with mold of fungus. That is why it is considered a occupational disease. The contamination happens with traumatic inoculation of material and some cases with inhalation of mold. When a human is infected, as well as other mammals or when cultivate at 37°C, it tums into the yeast form. Is common at tropical and moisture weather areas. Is found all around the world, and nowadays at Brazil, with cases related São Paulo, Rio de Janeiro and Rio Grande do Sul. At Rio de Janeiro, sporothricosis are related like endemic disease, where the etiologic agent is transmitted by cats to men. Sporothricosis is a chronic, polimorfic disease, and with form granulamatosa. The clinical type and the pathogenesis depend on the way of inoculation and the response of immunological system of the host. The defense and suscebility of the organism, as dissemination of infection with this fungi is not yet totally known. The patient\'s health state contributes directly to the disease, as much as alcoholism, diabetes and immunity deficiency and the extensive use of immunosuppressive drugs are factors that can bring to harder infection. Here, we studied in vitro the interaction of S. schenckii with murine macrophages. Using phagocytosis assay, ELISA, dosage of oxide nitric, CFU assay, flow citometry and RT-PCR were analysed activity of macrophages. We produced some results in order to better understand the response of defense with this disease. In the present study, we conclude that antibodies generated in mice by infection with S. schenckii were capable to promote phagocytosis of fungus. Also, when macrophages and yeast were incubated with laminarin or mannan, we had significant decrease in production of TNF-α, showing that receptors dectin-l and manose, respectively, are related with production this cytokine. The antibodies of mice infected with S. schenckii, also produced hard level this cytokine showing that entrance of fungus by receptor Fc can produce TNF- α. Besides, we verify that phagocitosys of yeast, with sera of infected mice, induct hard production of IL-10. When we analysed the expression of genes of macrophages, we observed hard expression of TLR-2 and MyD-88, when yeast were present. However, the expression of iNOS by macrophages, was more hard with yeast only when sera was present, before 72 hours. Similarly, when yeast were opsonized by sera, we had increase of production of NO after 72 hours of incubation. After, macrophage were capable to destruct yeast phagocyted when sera was present.

TITLE: IN VIVO ANTRAL FOLLICLE WALL BIOPSY: A NEW RESEARCH TECHNIQUE TO STUDY OVARIAN FUNCTION USING THE HORSE AS A MODEL Abstract Understanding hormone and growth factor receptor expression in the follicular wall associated with the intrafollicular milieu and systemic endocrine aspects in vivo is crucial for a complete discovery of mechanisms behind folliculogenesis in different species. Furthermore, the comprehension of the interrelationships existent among the preovulatory follicle (POF) and corpus luteum (CL) diameter and blood flow, and progesterone (P4) production during different seasons of the year are warranted to optimize fertility in different species. In this dissertation, four studies in mares are presented with the following general objectives: (i) develop and validate a minimally invasive in vivo technique to obtain antral follicle wall biopsy (FWB) and follicular fluid (FF) samples simultaneously and repeatedly from the same individuals, during different stages of follicle development in different seasons of the year; (ii) use the FWB technique to elucidate and compare the expression pattern of different receptors and proteins involved in folliculogenesis and ovulatory mechanisms during different seasons of the year; and (iii) use B-mode and color Doppler ultrasonography techniques to characterize POF and CL diameter and blood flow during two transitional ovulatory seasons (spring and fall), and elucidate the interrelationships among these variables with systemic P4 concentration. In the first study (Chapter 2), two experiments were conducted; in Experiment 1 (ex vivo), a proof of concept study was developed to check the feasibility of using the biopsy forceps to harvest FWB samples. Ovaries of six slaughtered mares were used, and each follicle was sampled using two techniques: biopsy forceps and scalpel blade (control). In Experiment 2 (in vivo), FWB and FF samples from growing follicles during various developmental stages were harvested using a novel FWB transvaginal ultrasound-guided technique. In the second study (Chapter 3), antral follicle wall and FF samples were obtained from 10−, 20−, and 30−mm follicles during spring anovulatory (SAN) and spring ovulatory (SOV) seasons. Samples were collected in vivo from the same mares in both seasons, with the aims to study and compare the expression of proliferative, angiogenic, and pro-/anti-apoptotic receptors and proteins in the follicle wall among three follicle classes, and between the SAN and SOV seasons. In the third study (Chapter 4), FWB and FF samples were collected in vivo from dominant growing follicles during the SAN, SOV, summer (SU), and fall ovulatory (FOV) seasons, to study the expression patterns of different receptors and proteins involved in follicle development. In the fourth study (Chapter 5), the temporal relationships among diameter and blood flow of the POF and CL, and systemic P4 concentration during spring and fall transitional ovulatory seasons were studied. The main findings of the present studies were: (i) the histological appearance, organization, and thickness of the follicle wall layers (i.e. granulosa, theca interna, and theca externa) were not influenced by the harvesting techniques (biopsy forceps vs. scalpel blade); (ii) the overall recovery rates of FWB and FF samples were 97% and 100%, respectively. However, the success rate of obtaining samples with all layers of the follicle wall and clear FF varied according to follicle size; (iii) follicles of the SOV season had a higher expression of proliferative, angiogenesis, and anti-apoptotic proteins compared to the SAN season; (iv) follicles of the FOV season had lower expression of proliferative, angiogenesis, and luteinizing hormone receptors; and (v) positive correlations were observed among POF and CL parameters, and P4 concentration. In conclusion, results demonstrated that the novel in vivo FWB technique developed in this study can be repeatedly and simultaneously used to provide sufficient FWB and FF samples for various cellular and molecular studies without jeopardizing the ovarian function in mares. Furthermore, results presented herein showed that several proliferative, angiogenic, and pro-/anti-apoptotic proteins are differentially expressed within different layers of the antral follicle wall among different seasons of the year. Finally, results demonstrated that larger and well-vascularized POFs have the potential to produce larger CLs with greater blood flow, and subsequently greater P4 systemic levels.

Sporothrix shenckii, um fungo dimórfico, termo-dependente, é encontrado na natureza associado com matéria orgânica em decomposição, como, espinhos, folhas secas, madeira e também na água. Este fungo é o agente causador da esporotricose, que ocorre com maior freqüência em pessoas que trabalham com o solo e vegetais contaminados com os esporos do fungo, sendo considerada uma doença ocupacional. A contaminação acontece com a inoculação traumática do material contaminado e em alguns casos por inalação dos esporos. Quando infecta o organismo humano, bem como outros mamíferos ou quando cultivado a 37°C, passa para a forma de levedura. É comum em áreas de clima tropical e subtropical, com alta umidade. Apresenta distribuição mundial, sendo que, atualmente no Brasil, são relatados casos em São Paulo, Rio de Janeiro e no Rio Grande do Sul. No Rio de Janeiro, a esporotricose tem sido relatada como doença endêmica, onde o agente etiológico é transmitido de gatos para homens. A esporotricose apresenta caráter crônico, polimórfico e com formação granulamatosa. As formas clínicas e a patogênese dependem do sítio de inoculação do fungo e da resposta imunológica do hospedeiro. O mecanismo de defesa e suscetibilidade do organismo, bem como a disseminação da infecção por este fungo ainda não são totalmente conhecidos. O estado de saúde do paciente contribui diretamente para o estabelecimento da doença, sendo que, alcoolismo, diabete, imunidade comprometida e uso extensivo de drogas imunosupressoras são fatores que podem predispor à infecções severas. Neste trabalho estudamos in vitro a interação do S. schenckii com macrófagos murinos. Por meio de técnicas de fagocitose, ELISA, dosagem de óxido nítrico, viabilidade de leveduras, citometria de fluxo e RT-PCR foram realizados ensaios da atividade de macrófagos. Desta forma foram gerados alguns dados para o melhor entendimento da resposta de defesa nesta doença. Nos estudos realizados, concluímos que os anticorpos gerados nos camundongos pela infecção por S. schenckii foram capazes de induzir a fagocitose do fungo. Também, após a fagocitose das leveduras pelos macrófagos, incubados com laminarina ou manana, houve inibição significativa da produção de TNF-α, indicando que os receptores dectina-l e manose, respectivamente, estão relacionados à produção desta citocina. O soro, contendo os anticorpos do animal infectado, também induziu forte produção desta citocina, mostrando que a entrada do fungo via receptor Fc pode também induzir a produção dessa citocina. Verificamos ainda que a fagocitose de leveduras, pelos macrófagos, na presença de soro de camundongo infectado, induziu alta produção de IL-10. Quando analisamos a expressão de genes presentes nos macrófagos, observamos forte expressão de TLR-2 e MyD-88, na presença das leveduras. Já a expressão de iNOS pelos macrófagos, observamos que foi maior na presença do fungo somente quando as leveduras foram incubadas com o soro, após o período de 72 horas. Em seguida, ao dosar a produção de Óxido Nítrico (NO), verificamos no período de 3 dias de incubação, que houve redução significativa na concentração de NO produzida pelos macrófagos, quando estes foram incubados com o fungo. Porém, quando as leveduras foram opsonizadas pelo soro, houve aumento da produção de NO após 72 horas de incubação. Outra verificação importante foi que os macrófagos foram capazes de destruir as leveduras fagocitadas na presença de soro. / Sporothrix schenckii is a dimorfic fungus, term-dependent, find in nature associated with organic material at decomposition, like thorns, wheat leaves, wood and water. This is the fungi agent that causes sporothricosis, which happens with more frequency with people that work with land and green, contaminated with mold of fungus. That is why it is considered a occupational disease. The contamination happens with traumatic inoculation of material and some cases with inhalation of mold. When a human is infected, as well as other mammals or when cultivate at 37°C, it tums into the yeast form. Is common at tropical and moisture weather areas. Is found all around the world, and nowadays at Brazil, with cases related São Paulo, Rio de Janeiro and Rio Grande do Sul. At Rio de Janeiro, sporothricosis are related like endemic disease, where the etiologic agent is transmitted by cats to men. Sporothricosis is a chronic, polimorfic disease, and with form granulamatosa. The clinical type and the pathogenesis depend on the way of inoculation and the response of immunological system of the host. The defense and suscebility of the organism, as dissemination of infection with this fungi is not yet totally known. The patient\'s health state contributes directly to the disease, as much as alcoholism, diabetes and immunity deficiency and the extensive use of immunosuppressive drugs are factors that can bring to harder infection. Here, we studied in vitro the interaction of S. schenckii with murine macrophages. Using phagocytosis assay, ELISA, dosage of oxide nitric, CFU assay, flow citometry and RT-PCR were analysed activity of macrophages. We produced some results in order to better understand the response of defense with this disease. In the present study, we conclude that antibodies generated in mice by infection with S. schenckii were capable to promote phagocytosis of fungus. Also, when macrophages and yeast were incubated with laminarin or mannan, we had significant decrease in production of TNF-α, showing that receptors dectin-l and manose, respectively, are related with production this cytokine. The antibodies of mice infected with S. schenckii, also produced hard level this cytokine showing that entrance of fungus by receptor Fc can produce TNF- α. Besides, we verify that phagocitosys of yeast, with sera of infected mice, induct hard production of IL-10. When we analysed the expression of genes of macrophages, we observed hard expression of TLR-2 and MyD-88, when yeast were present. However, the expression of iNOS by macrophages, was more hard with yeast only when sera was present, before 72 hours. Similarly, when yeast were opsonized by sera, we had increase of production of NO after 72 hours of incubation. After, macrophage were capable to destruct yeast phagocyted when sera was present.