Orientadores: Gilberto de Nucci, Elen Cristina Teizem Landucci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T21:27:11Z (GMT). No. of bitstreams: 1 Ferreira_Tatiane_M.pdf: 838991 bytes, checksum: fcfc9182e3cb79df03511f3867236e61 (MD5) Previous issue date: 2008 / Resumo: A habilidade do veneno bruto de Lachesis muta muta e da fosfolipase A2 básica (LmTX-I) de induzir aumento da permeabilidade microvascular na pata e pele de ratos foi investigada nesse trabalho. O veneno bruto ou LmTX-I foi injetado subplantar ou intradermicamente e o edema foi medido após intervalos de tempo específicos. O volume da pata foi medido usando um hidropletismômetro, enquanto o extravasamento na pele foi medido através do acúmulo de albumina humana marcada com 125I (injetada previamente i.v.) nos sítios de pele. A liberação de histamina dos mastócitos de ratos foi medida por espectrofluorometria. O veneno bruto (0,3-3 µg/pata) ou LmTX-I (0,1-1 µg/pata) induziu edema de pata de maneira dose-dependente. A injeção intradérmica do veneno bruto (0,003-10 µg/sítio) ou LmTX-I (0,003-0,3 µg/sítio) na pele dorsal também resultou em extravasamento de proteínas plasmáticas de maneira dose-dependente. O extravasamento plasmático induzido pelo veneno bruto foi significativamente diminuído pelo tratamento com mepiramina (antagonista de receptor H1, 6 mg/kg), ciproeptadina (antagonista H1 e 5-HT2, 2 mg/kg), LNAME (inibidor não seletivo da síntese de óxido nítrico, 100 nmol/sítio), SR140333 (antagonista de receptor NK1, 1 nmol/sítio), icatibant (antagonista de receptor B2, 0,6 mg/kg) e indometacina (inibidor não seletivo das cicloxigenases, 5 mg/kg), mas não pelo tratamento com PCA4248 (antagonista de receptor de PAF, 5 mg/kg). O extravasamento na pele induzido pela LmTX-I foi significativamente inibido pela ciproeptadina, mepiramina, indometacina e PCA4248, enquanto que L-NAME, SR140333 e icatibant não tiveram efeito. Tanto o veneno bruto quanto a LmTX-I induziram a liberação de histamina de mastócitos peritoneais de ratos de maneira concentração-dependente. Em conclusão, o veneno de L. m. muta e a LmTX-I aumentam a permeabilidade microvascular por mecanismos envolvendo a ativação de mastócitos e a formação de metabólitos do ácido araquidônico. A resposta induzida pelo veneno bruto também envolve liberação de substância P, bradicinina e óxido nítrico enquanto a resposta induzida pela LmTX-I conta com a participação do PAF. / Abstract: The ability of crude venom and a basic phospholipase A2 (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in the rat paw and skin has been investigated. Crude venom or LmTX-I were injected subplantarly or intradermically, after which oedema was measured at selected times thereafter. Paw volume was measured using a hydropletismometer, whereas skin extravasation at the skin sites was measured as accumulation of i.v. injected 125I-human serum albumin. Histamine liberation from rat mast cell was measured spectrofluorometrically. Crude venom (0.3-3 µg/paw) or LmTX-I (0.1-1 µg/paw) induced a dose-dependent rat paw oedema. Intradermal injection of crude venom (0.03-10 µg/site) or LmTX-I (0.003-0.3 µg/site) in the dorsal skin also resulted in dosedependent plasma extravasation. Crude venom-induced plasma extravasation was significantly inhibited by the histamine H1 receptor antagonist mepyramine (6 mg/kg), the histamine/5-hydroxytriptamine antagonist cyproheptadine (2 mg/kg), the nitric oxide synthesis inhibitor N?-L-nitro-arginine methyl ester (L-NAME; 100 nmol/site), the tachykinin NK1 receptor antagonist SR140333 (1 nmol/site), the bradykinin B2 receptor antagonist Icatibant (0.6 mg/kg) and the cyclooxygenase inhibitor indomethacin (5 mg/kg). The platelet-activating factor receptor antagonist PCA4248 (5 mg/kg) had no significant effect. LmTX-I-induced skin extravasation was significantly inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while L-NAME, SR140333 and Icatibant had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat peritoneal mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving mast cell activation and both had arachidonic acid metabolites participation. Crude venom-induced responses also involves substance P, bradykinin and nitric oxide release, whether LmTX-I-induced response involves PAF. / Mestrado / Mestre em Farmacologia

Alopecia areata (AA) is a chronic inflammatory disease of hair follicles manifesting as patchy areas of hair loss on the scalp and body. Development of AA is associated with pen- and intra-follicular inflammation of anagen stage hair follicles, primarily by CD4+ and CD8+ cells. We hypothesized that if cell-mediated cytotoxicy against hair follicles is to be a component of the hair loss disease mechanism, increased expression of genes and products typical of cytotoxic cells, as well as increased apoptosis activity within affected hair follicles, would be expected to occur in the lesional skin compared to the normal skin. Furthermore, we studied gene expression levels of multiple cytokines and characteristic chemokines, using the C3FI/HeJ mouse model of AA. mRNA expression levels of granzyme A, granzyme B, perform Fas, Fas ligand, TNF-cL, TNF-aRl and R2, TRAIL, TRAILR, TRAMP, Thi-, Th2-, and Th17-associated cytokines, as well as multiple chemokines were compared between the skin, draining lymph nodes, thymus and spleens of normal and AA-affected mice using quantitative reverse transcriptase PCR. FasL, granzyme A, granzyme B, pro- and anti-inflammatory cytokines were all highly up-regulated in the skin of AA-affected mice. Immunohistochemical studies of the skin revealed that, although greater numbers of granzyme B and FasL expressing cells were present in AA affected skin, the cells were morphologically diffusely distributed and not exclusively located within the focal pen- and intrafollicular infiltrate. The majority of these cells were further characterized as mast cells, which were also found in substantially greater numbers in the skin of mice with AA compared to their normal haired controls. Almost no perform expressing cells were identified in AA affected mouse skin and TUNEL staining suggested relatively limited apoptosis activity in hair follicle keratinocytes. In conclusion, while granzymes and FasL may play important roles in disease development, the profiles and patterns of expression are not consistent with direct cell-mediated cytotoxic action against the follicular epithelium in chronic mouse AA. Potentially, hair growth inhibiting cytokines may play a more dominant role in AA development than previously thought. Furthermore, mast cells, with their increased presence around hair follicles in the AA affected mouse skin and their ability to express granzyme B and FasL, are suggested as potential key players in the pathogenesis of AA. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Gout is characterized by the deposition of monosodium urate (MSU) crystals. Despite being one of the most painful forms of arthritis, gout and the mechanisms responsible for its acute attacks are poorly understood. In the present study, we found that MSU caused dose-related nociception (DE50=0.04 (0.01-0.11) mg/paw) and edema (DE50=0.08 (0.04-0.16) mg/paw) when injected into the hind paw of rats. Treatment with the selective TRPV1 receptor antagonist SB366791 largely inhibited nociceptive and edematogenic responses to MSU. Moreover, the desensitization of capsaicin-sensitive afferent fibers as well as the pretreatment with the tachykinin NK1 receptor antagonist RP 67580 also significantly reduced MSU-induced nociception and edema. Once MSU was found to induce mast cell stimulation, we investigated the participation of these cells on MSU effects. Prior degranulation of mast cells by repeat treatment with compound 48/80 decreased MSU-induced nociception and edema or histamine and serotonin levels in the injected tissue. Moreover, pretreatment with the mast cell membrane stabilizer cromolyn effectively inhibited nociceptive and edematogenic responses to MSU. MSU induced a release of histamine, serotonin and tryptase in the injected tissue, confirming mast cell degranulation Furthermore, the antagonism of histaminergic H1 and serotoninergic receptors decreased the edema, but not the nociception, of MSU. Finally, the inhibition of tryptase activity was capable of largely reducing either MSU-induced nociception or edema. Collectively, the present findings demonstrate that MSU produces a nociceptive and edematogenic response mediated by TRPV1 receptor activation and mast cell degranulation. / A gota é caracterizada pela deposição de cristais de urato monossódico (MSU) nas articulações. Apesar de ser um dos mais dolorosos tipos de artrite, os mecanismos responsáveis pela indução da dor durante os ataques agudos de gota são pouco entendidos. No presente estudo, objetivamos investigar o papel do receptor TRPV1 na nocicepção e edema induzidos por cristais de MSU em ratos. Assim, demonstramos que o MSU causa nocicepção (DE50=0.04 (0.01-0.11) mg/pata) e edema dependentes da dose (DE50=0.08 (0.04-0.16) mg/pata) quando injetado na pata dos ratos. O tratamento com o antagonista seletivo do receptor vanilóide TRPV1 SB 366791 inibiu significativamente as respostas nociceptiva e edematogênica causadas pelo MSU. De maneira semelhante, a dessensibilização de fibras aferentes sensíveis a capsaicina bem como o tratamento com o antagonista do receptor para taquicinina NK1 RP67580 também reduziram significativamente a nocicepção e o edema induzidos pelo MSU. Sabendo que estudos prévios demonstraram que MSU induz a estimulação de mastócitos, nós investigamos a participação destas células nos efeitos do MSU. A desgranulação prévia de mastócitos por tratamento repetido com o composto 48/80 reduziu a nocicepção e o edema induzidos pelo MSU assim como os níveis de histamina e serotonina no tecido injetado. Adicionalmente, o tratamento com o estabilizador de membrana de mastócitos cromolina, reduziu efetivamente as respostas nociceptivas e edematogênicas ao MSU. A administração de MSU induziu a liberação de histamina, serotonina e triptase no tecido injetado, confirmando a desgranulação mastocitária. Além disso, o antagonismo de receptores histaminérgicos H1 e serotoninérgicos, reduziram o edema, mas não a nocicepção causados pelo MSU. Finalmente, a inibição da atividade da triptase foi capaz de reduzir amplamente a nocicepção e o edema induzidos pelo MSU. Coletivamente, nossos resultados demonstram que o MSU produz uma resposta nociceptiva e edematogênica mediada pela ativação do receptor TRPV1 e pela desgranulação de mastócitos.

Mast cells are critical effectors of allergic disease that can be activated by numerous stimuli. We have examined mast cell control by the inflammatory cytokine, IL-33, as well as IgG. In the first study reported here, we found that the synthetic glucocorticoid, dexamethasone, potently and rapidly suppressed IL-33-induced cytokine production from murine bone marrow–derived and peritoneal mast cells, as well as human mast cells. Dexamethasone also antagonized IL-33-mediated enhancement of IgE-induced cytokine production and migration. Although dexamethasone had no effect on IL-33-induced phosphorylation of MAP kinases or NFκB p65 subunit, it antagonized AP-1 and NFκB-mediated transcriptional activity. Finally, intraperitoneal administration of dexamethasone completely abrogated IL-33-mediated peritoneal neutrophil recruitment and prevented plasma IL-6 elevation. These data demonstrate that steroid therapy may be an effective means of antagonizing the effects of IL-33 on mast cells in vitro and in vivo, acting partly by suppressing IL-33-induced NFκB and AP-1 activity. In the second study reported here, we found that Fcγ receptor crosslinkage activated the transcription factor Stat5B through a Fyn kinase-dependent pathway. We then showed that STAT5B is critical for IgG-induced cytokine production by mast cells but not macrophages. To expand these studies, we employed the K/BxN model of inflammatory arthritis, which has roles for mast cells and macrophages. In this model, Fyn or STAT5B deficiency only affected the arthritic flare that primarily depends on mast cell degranulation, without affecting the severity of the joint swelling. By contrast, Lyn kinase deficiency significantly exacerbated arthritis. These studies indicate a clinically relevant, lineage-restricted role for the Lyn/Fyn-STAT5 cascade. Collectively, our work demonstrates that mast cell activation by diverse stimuli can be suppressed by steroid intervention and selectively targeted by disrupting kinase-transcription factor pathways.

Abstract Both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. There are several hypothetical mechanisms for drug-induced gingival overgrowth, such as the influence of genetic predisposition, alterations in gingival tissue homeostasis, especially in the function of fibroblasts, and drug-induced action on growth factors. Clinical studies have also shown that, those with poor oral hygiene status drug-induced gingival overgrowth is more prevalent and severe than those with good oral hygiene status. The working hypothesis was that immunosuppressive medication and/or nifedipine medication affects inflammatory cell profile and mitotic activity of keratinocytes in human overgrown gingiva. We studied gingival samples, collected from nifedipine-medicated cardiac outpatients and immunosuppression-medicated organ-transplant recipients. Patients were placed into four groups: 1) the immunosuppression group, patients receiving cyclosporin-A (CsA), azathioprine (AZA) and prednisolone (Pred) 2) the immunosuppression plus nifedipine group, patients receiving CsA, AZA, Pred. and nifedipine 3) the nifedipine group patients receiving only nifedipine and 4) the non-medicated control group. All of the samples related to moderate to severe degrees of gingival overgrowth, covering half to two thirds of the clinical crown. The aim of the study was to investigate the occurrence of Langerhans cells, macrophages, mast cells and mitotic activity of keratinocytes in human drug-induced overgrown gingiva, and consequently to assess their possible role in the pathogenesis of drug-induced gingival overgrowth. We found that immunosuppressive medication increased the numbers of reparative macrophages (RM3/1) and decreased the numbers of tryptase- and chymase-positive mast cells (MCTC) cells. We have also shown that immunosuppressive and nifedipine medication decreased the numbers of Langerhans cells (CD1a) and increased the numbers of 27E10-macrophages parallelly. Additionally we found increase in the mitotic activity of gingival keratinocytes and even two-fold thickening of gingival epithelium in immunosuppressive and nifedipine medication-induced gingival overgrowth as compared with healthy gingiva. Immunosuppressive medication activated gingival epithelium (27E10 expression in gingival keratinocytes) more than nifedipine medication. In conclusion, our results suggest that gingival overgrowth among immunosuppressive- and nifedipine-medicated patients is related to alteration of tissue homeostasis. First, this suggestion is supported by changes found in the numbers of cells that directly affect connective tissue turnover, e.g. reparative macrophages (RM3/1) and mast cells. Changes in the numbers of these cells could alter the cytokine- and growth factor-profile, which affects fibroblast function. Secondly, we found changes in the numbers of cells involved in regulation of inflammation, e.g. Langerhans cells and monocytes as compared with healthy controls. Immunosuppressive medication could directly activate gingival keratinocytes. We suggest that our findings mainly reflect the effects of immunosuppressive medication, but the role of inflammation cannot be excluded. The changes observed above represent differences of the pathogenesis of drug-induced gingival overgrowth between immunosuppressive and nifedipine medication. It must be however remembered that drug-induced gingival overgrowth is a result of multicausal intrinsic and extrinsic factors. Age, gender, concomitant medication with multiple drugs, plaque accumulation, and genetic disposition are additional risk factors. The abnormal distribution of specific immune system cell subpopulations does not alone prove a functional relationship to gingival overgrowth.

O mundo do petróleo envolve investimentos de bilhões de dólares por ano, sendo que falhas e acidentes com sondas de perfuração/produção de poços de petróleo podem resultar em severas conseqüências econômicas e judiciais as empresas responsáveis pela operação destes equipamentos. Atualmente no Brasil a maior parte das sondas de produção terrestre está em operação há mais de três décadas e não há um levantamento ou acompanhamento estatístico dos acidentes operacionais ocorridos durante a atividade de intervenção em poços de petróleo ou gás com sondas de produção terrestres. Entretanto, acidentes com estes equipamentos são freqüentes em campos brasileiros, porém devido à falta de uma fiscalização rígida, muitos destes acidentes são negligenciados. Este estudo visa à elaboração de um modelo pelo método de elementos finitos para avaliação dos limites operacionais de estruturas de mastros de sondas de produção terrestres. O trabalho foi elaborado a partir dos requisitos e recomendações técnicas da Norma API Spec 4F - 3ª edição: 2008. O estudo apresentou o comportamento estrutural do mastro da sonda de produção terrestre após alguns anos em operação, a partir de um estudo de engenharia reversa em campo. Foi realizado um levantamento das características geométricas, dimensionais e tipos de materiais utilizados na fabricação da estrutura. Após o levantamento em campo, foram analisados todos os requisitos e requerimentos técnicos da norma API 4F aplicáveis à realidade da operação destes equipamentos em campos brasileiros. Através do modelo de elementos finitos foram apresentados em forma de gráficos os limites operacionais de carregamentos simples e combinados que a estrutura da sonda pode ser submetida. Também é apresentado um estudo comparativo entre a carga de içamento estático especificada originalmente pelo fabricante e o valor atual que a estrutura pode ser submetida, bem como as regiões mais críticas do mastro. / The world oil involves investiments of billions dollars per year, however, failures and accidents with drilling rigs / well servicing rigs mast of oil Wells can result in severe consequences economics and legals to companies responsibles by operation this equipments. Currently in Brazil most onshore rigs are in operation more than three decades and there isn´t survey or statistics monitoring of the operational accidents ocurred during well servicing activity in well oil or gas with onshore rigs masts. However accidents with this equipments are frequents in Brazilian fields, but due absence of a rigid inspection, many of these accidents are ignored. This study aimed at developing a model by the Finite Element Method to evaluate the structural operational limits of onshore well servicing rigs masts. The dissertation was drafted from the technical requirements and recommendations of API Spec 4F - 3rd 2008 Edition Standard. The study showed the structural behavior of onshore well servicing probe mast structure after a few years in operation, from a reverse engineering study in the field. A survey of geometric features, dimensions and types of materials used in the structure manufacturing was conducted. After the field survey, all the API 4F Standard technical requirements, applicable to this equipment operational reality in Brazilian fields were analyzed. By the Finite Element Model, the operational limits of single and combined loads that the probe structure can be subjected were presented in graph form. A comparative study between static hoisting load originally specified by the manufacturer and the current value that the structure can be submitted was also presented, as well as the most critical regions of the mast.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22686号 / 医博第4630号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Wildlife managers use models to aid in predicting high risk areas for human and black bear (Ursus americanus) interactions (HBI). These tools help managers implement management strategies to minimize HBI. Over 3,000 incidents of HBI were compiled from management reports at Great Smoky Mountains National Park (GSMNP) during 1998-2011, a park with 9-10.2 million visitors per year and a black bear population of about 1,600 bears. We used data from bear management reports along with annual visitor use, mast and bear abundance data to develop a series of generalized linear models to assess the spatial and temporal factors affecting HBI. Although HBI occurred throughout the GSMNP, 50% of all HBI occurred in five areas. The best predictor variables of HBI across four subsets of models included interaction between mast production and number of park visitors, month, vegetation cover, visitor activity, and bear abundance. Although there was not a clear relationship between visitor use and mast abundance, the number of park visitors was always relatively high and HBI increased substantially in poor mast years. HBI was more frequent during summer months when park visitation rates and more people and food were present overnight in frontcountry and backcountry camping areas. Over 43% of HBI in hemlock forests were serious. Bear abundance data were not a strong predictor of HBI, and bear bait stations may not provide a sensitive index to bear abundance. GSMNP uses different strategies for managing HBI to protect visitors and bears. In 1991, bear proof waste disposal containers and food storage devices were placed in camping and picnic areas. In combination with aversive conditioning, HBI decreased in some areas of the park. We recommend that proactive bear management programs including education, enforcement of park regulations, and aggressive aversion conditioning of bears be implemented at the identified HBI high risk areas to provide a safer environment for both people and bears in GSMNP.

4 Abstract Mast cells are involved in variety of immunological processes, but they are mostly known for their role in allergy and asthma. As asthma and allergy are serious diseases with spreading tendency during last decades, mast cells are subject of intensive research. It is expected that studies of mast cell signalling pathways will contribute to our understanding of the nature of these diseases and help to design efficient treatment strategies. In an attempt to identify genes responsible for asthma disease, genome-wide screening methods have been currently applied. Using these methods, mutations in ORMDL3 (Orosomucoid1-like) protein were found out as a high risk asthma factor. ORMDL3 is a member of evolutionary conserved ORMDL family, comprising in mammals also of ORMDL1 and ORMDL2. Physiological function of these proteins is poorly understood and it has not been studied in mast cells. We decided to study the role of ORMDL proteins in mast cells. Lentiviral delivery system was optimised for generation of stable knock-downs (KD) of all three members of the ORMDL family in primary mast cells. The ORMDL gene expression was measured by improved qPCR (quantitative PCR) reaction buffers. We found that all ORMDL genes are expressed in mast cells in order ORMDL3 > ORMDL2 > ORMDL1. Next, we investigated the...

Mast cells (MCs) are long-lived immune cells, which were reported to play an important role in initiating innate and adaptive immune responses against various infections. MCs accumulate in high numbers in the stroma and at the invasion front of various human cancers, suggesting a possible contribution by MCs to tumour growth. Experimental studies using crosses of MC-deficient Kit-mutant mouse strains with mouse models of epithelial cancers have provided evidence for important MC tumour-promoting functions. However, the complex alterations of the immune system that characterize Kit-mutant mice in addition to their MC deficiency, limit the interpretation of these findings. Numerous key observations made in Kit mutant mice were not reproduced in novel, Kit-independent mouse models of MC deficiency. Thus, the impact of MCs on tumour biology remains unclear. The aim of this study is to clarify the contribution of MCs to the biology of Human papilloma virus (HPV)-induced skin cancer in a Kit-independent mouse model of MC deficiency. In K14-HPV16 transgenic mice, HPV oncogenes are constitutively expressed in the epidermis resulting in epidermal hyperplasia with 100% penetrance and squamous cell carcinoma in about 50% of the animals. A cross to a Kit-mutant line suggested that MCs are important tumour promoters in this model. We crossed K14-HPV16 mice to M5Cre R-DTA line, in which MCs are constitutively depleted with high efficiency and selectivity. Unexpectedly, the loss of MCs neither affected keratinocyte proliferation indices nor altered keratinocyte apoptosis at any stage of HPV-induced neoplasia. Furthermore, the loss of MCs did not result in any detectable changes in composition and gene expression of the inflammatory hematopoietic cell infiltrate in the tumour stroma. This shows that, contrary to current belief, MCs have no important function in orchestrating the tumour micro milieu. In keeping with this finding, MC deficiency resulted in no detectable difference in the incidence growth or grading of SSC in K14-HPV16 transgenic mice. Collectively, these results show that, despite their high density in HPV-induced neoplasia, MC have no role in cancerogenesis or neoplastic progression in the K14-HPV16 mouse model. Our findings also emphasize the importance of novel Kitindependent mouse models in the investigation of MC in vivo functions.