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No description available.

A paracoccidioidomicose (PCM) é uma doença granulomatosa sistêmica, causada por Paracoccidioides spp., (P. brasiliensis e P. lutzii), geograficamente, limita-se a América Latina com as áreas endêmicas estendendo-se desde o México até a Argentina, constituindo uma das micoses sistêmicas de maior incidência na região, afetando principalmente trabalhadores rurais. O maior número de pacientes com PCM tem sido reportado principalmente no Brasil, Colômbia e Venezuela. A incidência real desta micose encontra-se subestimada no Brasil e pouco se conhece em relação a nova espécie descrita - P. lutzii. A maioria dos estudos em P. lutzii foram focados em genética, especiação e na geração de novos antígenos para melhorar a especificidade e sensibilidade dos testes sorológicos. Atualmente, as preparações antigênicas tradicionais, preparadas a partir de isolados de P. brasiliensis, são ineficientes. Raros são os trabalhos focados na biologia de P. lutzii e nos fatores de virulência que podem ser comparados com P. brasiliensis nos modelos experimentais. A nossa proposta de estudo foi avaliar alguns aspectos in vitro e in vivo relacionados com a patogenicidade e destacamos: a fagocitose e a morte intracelular de P. lutzii por macrófagos, peritoneais, de camundongos Knockouts (KO) e selvagens para PRRs (TLR2, TLR4 e Dectina) e ativadores intracelulares (MyD88 e NALP3). Paralelamente a este estudo, animais foram infectados com leveduras de P. lutzii e comparados com os modelos de infecção já estabelecidos com leveduras (Pb18) e conídios (ATCCPb60855) de P. brasiliensis. Nossos dados indicam que similar ao que ocorre com P. brasiliensis a fagocitose de P. lutzii depende de TLR2, TLR4 e Dectina- 1, resultados semelhantes também foram observadas na expressão de moléculas envolvidas na co-estimulação e a apresentação de antígenos (MHC II, CD80 e CD86). Contudo, a morte intracelular de leveduras de P. lutzii é claramente dependente de TLR4, e a produção de citocinas IL-6, MIP-2, IFN- e IL-12p40 são importantes para o controle das leveduras pelos macrófagos. No modelo experimental de P. lutzii, camundongos machos C57BL/6 (6-7 semanas) foram infectados intratraquealmente como 1x106 leveduras viáveis do isolado de P. lutzii Pb01. Encontramos duas fases da doença, a primeira de 0 hora até 2 a 4 semanas pós-infecção, e a segunda de 4 até 12 semanas. As leveduras parecem ser contidas na primeira semana de infecção e posteriormente não encontramos leveduras nos macerados de pulmão, diferente do modelo de BALB/c infetado com conídios de ATCC-Pb60855 no qual as UFC são recuperadas até a semana 16 pós-infeção. Como relação aos níveis de citocinas, encontramos que na lavagem broncoalveolar e macerado de pulmão um perfil misto Th1/Th2 porém, marcado por citocinas próinflamatórias no primeiro período e citocinas regulatórias tipo Th2 no segundo período (IL-12p70, IL-23, IL-10); similar ao descrito nos modelos de P. brasiliensis infectados tanto com conídios como com leveduras. No entanto, no primeiro período da doença, em camundongos C57BL/6, parece ter uma carga inflamatória maior que reflete nas citocinas que mantém seus níveis até o período crônico: TNF-alfa, MIP-2 e GM-CSF está última, regulada positivamente tanto em experimentos in vitro como in vivo. Também observamos que a partir das 48horas pós-infecção encontramos níveis aumentados de IL-12p70 até o período crônico onde junto com a IL-23 parecem ser as responsáveis pela diminuição da infecção no período tárdio. Esta é a primeira vez que se descreve um modelo experimental com P. lutzii (isolado Pb01) indicando o perfil imunopatológico com pequenas diferenças comparados ao P. brasiliensis porém, de importância na patogenicidade da doença auxiliando a compreender as diferentes formas da doença no modelo experimental / Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by Paracoccidioides spp. (P. brasiliensis and P. lutzii), geographically, is limited to Latin America with endemic areas from Mexico to Argentina, as one of the systemic mycoses with the highest incidence in the region, mainly affecting rural workers. The largest number of patients with PCM has been mainly reported in Brazil, Colombia and Venezuela. The true incidence of this mycosis is underestimated in Brazil and little is known about the new species described - P. lutzii. Most studies in P. lutzii were focused on genetics, speciation and the generation of new antigens to improve the specificity and sensitivity of serological tests. Currently, traditional antigenic preparations, prepared with isolates of P. brasiliensis, are inefficient. There are few studies focused on P. lutzii biology and virulence factors that can be compared with P. brasiliensis in experimental models. Our study aimed to evaluate some in vitro and in vivo aspects related to pathogenicity: phagocytosis and intracellular killing of P. lutzii by peritoneal macrophages from knockouts (KO) for PRRs (TLR2, TLR4 and Dectin) and intracellular activators (MyD88 and NALP3). In addition, animals were infected with P. lutzii yeast and compared with the well-established models of infection with yeast cells (Pb18) and conidia (ATCC Pb60855) from P. brasiliensis. Our data indicate that similarly to what happens with the phagocytosis of P. brasiliensis, P. lutzii phagocytosis is dependent on TLR2, TLR4 and Dectin-1. Other molecules, involved in co-stimulation and presentation of antigens such as MHC II, CD80 and CD86 were also shown to participate in the P. lutzii-host interaction. However, intracellular killing of P. lutzii yeast cells was clearly dependent on TLR4, and the production of cytokines as IL-6, MIP-2, IFN- and IL-12p40 were important for the control of the yeast by macrophages. In the experimental model of P. lutzii, male C57BL/6 mice (6-7 weeks) were infected intratracheally with 1x106 viable yeasts of the isolate Pb01like. We found two phases of the disease, the first from the inoculation to 2 or 4 weeks after infection and the second from 4 to 12 weeks. Yeast appear to be contained within the first week of infection and subsequently are also absent from macerated lung, differently from the model of BALB/c mice infected with ATCC Pb60855 conidia in which CFUs were detected up to week 16 post-infection. We found a mixed Th1/Th2 pattern (IL-12p70, IL-23, IL-10) in bronchoalveolar lavage and lung, with the predominance of proinflammatory cytokines in the first phase and predominance of regulatory Th2 cytokines in the second phase, reproducing findings of P. brasiliensis infection models produced with both conidia and yeast. However, in the first period of the disease in C57BL/6 mice there was a higher inflammatory burden, reflected by the high cytokine levels (TNF-alpha, MIP-2 and GM-CSF), the latter in particular because it was positively regulated both in vitro and vivo), that persisted through the chronic period. We also observed that starting from 48 hours postinfection to the chronic period there were increased levels of IL-12p70, which together with IL-23 appeared to be responsible for the reduction of infection in the late period. This is the first time that an experimental model with P. lutzii (Pb01) is described, showing an immunological profile with only slight differences compared to the P. brasiliensis model. The present study details important aspects of the pathogenesis of the disease due to different species of Paracoccidioides and helps to understand the different forms of presentation in experimental models

First study determined the effects of genetic deletion of nNOS on the levels of spontaneous apoptosis in brain of young-adult (2-3 months) and aged (12-18 months) mice, using nNOS-knockout mice with age-matched B6129SF2/J mice as wild-type control. The results indicate that aging resulted in 11-fold increase in levels of apoptotic-DNA-fragmentation in B6129SF2/J mouse brain. nNOS-knockout mice demonstrated dramatic (72-fold) increases in levels of apoptotic-DNA-fragmentation in young-adult, but not aged, brains. Aging resulted in decreased number of nNOS-positive cells, increased number of iNOS-positive cells and no change of eNOS-positive cells in control mice. The data suggest that nNOS may serve an anti-apoptotic/neuroprotective role in young-adult mouse brain. However, because of diminished nNOS and increased iNOS with aging, this neuroprotective effect may become less effective in aged mice. / Fourth study showed that new microchip-electrophoresis-technology can be successfully used to identify and quantify levels of apoptosic-DNA-fragments in brain slice cultures, similar to our previous studies with CE-LIF. Because of the much greater throughput of microchip-electrophoresis-system, compared to CE-LIF, this new technology should help accelerate the progress of apoptosis research. / In second study, apoptotic effects of genetic deletion of either eNOS or iNOS were studied using young-adult (1-4 months) and aged (12-24 months) eNOS- or iNOS-knockout mice with age-matched C57BL/6J wild-type control mice. The data show that both young-adult and aged iNOS-knockout mice had dramatically (8- to 36-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially noticeable in hippocampus and medulla oblongata. Both young-adult and aged eNOS-knockout mice also had dramatically (18- to 35-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially in cerebral cortex, hippocampus and medulla oblongata. The data suggest that both iNOS and eNOS provide neuroprotective effects, helping to limit the extent of spontaneous apoptosis in brain of young-adult and aged mice. / Nitric oxide (NO) has either pro-apoptotic or anti-apoptotic effects on neuronal cells, depending on concentration of NO produced by different source of NO synthases (NOSs) including neuronal-NO-synthase (nNOS/NOS-1), inducible-NO-synthase (iNOS/NOS-2) or endothelial-NO-synthase (eNOS/NOS-3) and possibly age of the individual. The present study determines if genetic deletion of nNOS, iNOS or eNOS alters levels of aging-induced apoptosis in vivo and hydrogen peroxide (H2O2)-induced-apoptosis in organotypic brain slice cultures using NOS-knockout mice. The quantitative ultrasensitive techniques using capillary-electrophoresis with laser-induced-fluorescent detector (CE-LIF) and Cell-Death---Detection-ELISA were used as novel ways to accurately measure the levels of apoptotic-DNA-fragmentation. Expressions of different forms of NOSs were determined by immunohistochemical-staining. / Third study determined H2O2-induced apoptosis in hippocampal and cerebellar slices from young-adult (8-10 weeks) and aged (12-24 months) C57BL/6J control mice, as well as iNOS- and eNOS-knockout mice (determined by Cell-Death-Detection-ELISA measuring levels of apoptotic-DNA-fragmentation). The data show spontaneous onset of apoptosis occurred in both hippocampal and cerebellar slices during culturing, beginning at 24 hours and progressively increasing for 48--72 hours. Staurosporine (positive-control) and H2 O2 both caused time-dependent increases in apoptosis in both hippocampal and cerebellar slices, compared to time-matched controls. Lastly, genetic deletion of iNOS greatly reduced levels of spontaneous apoptosis in young hippocampus and aged cerebellum, suggesting iNOS had contributed to induction of spontaneous apoptosis. / Chow Wing Han Vivian. / "Dec 2005." / Advisers: Siew Boon Chew Cheng; Ray Ronald Fiscus. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6218. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 144-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

O conhecimento de que o exercício é benéfico na doença aterosclerótica é baseado principalmente em estudos epidemiológicos. O objetivo deste trabalho foi investigar se o treinamento físico preventivo ou terapêutico altera a evolução da placa aterosclerótica. Camundongos LDLr-/- com 16 semanas de vida foram separados em dois programas: preventivo e terapêutico. Animais do programa preventivo receberam dieta normal ou aterogênica por 14 semanas. O treinamento físico foi iniciado concomitantemente ao início da dieta. Animais do programa terapêutico receberam dieta normal ou aterogênica por 28 semanas. O treinamento físico foi iniciado após 14 semanas do início da dieta, com placas bem estabelecidas. O treinamento físico aeróbico moderado foi realizado em esteira rolante, por 60 min, 5 dias/sem, durante 14 semanas. Massa corporal, pressão arterial caudal e freqüência cardíaca foram registradas. Lipoproteínas plasmáticas foram separadas por FPLC e colesterol total foi dosado por métodos enzimáticos. Foram quantificados tamanho, conteúdo de gordura e de colágeno da placa por coloração de oil-red O e picro-sirius. Citocinas TNF-, IL-6 foram medidas por Elisa. MMP-9 plasmática foi medida por zimografia. Marcadores inflamatórios teciduais, MMP-9, CD40/CD40L e nitrotirosina, foram medidos na placa por imunohistoquímica. O treinamento físico não modificou o tamanho da placa, mas tornou a placa mais estável por aumentar o conteúdo de colágeno. O treinamento físico diminuiu o conteúdo de gordura da placa, os fatores de risco e o CD40 somente no programa preventivo. Nenhuma alteração foi notada nos marcadores inflamatórios circulantes e na expressão de MMP-9 e formação de nitrotirosina na placa aterosclerótica. / The knowledge that exercise exerts beneficial effects on atherosclerotic disease is mainly based on epidemiological studies. Our aim was to investigate the effect of preventive and therapeutic exercise programs on atherosclerotic plaque formation and development. Sixteen-week-old LDLr-/- mice were randomly divided into preventive and therapeutic programs. Preventive programs mice received normal or atherogenic diet for 14 weeks. Exercise training started at the same time of dieting. Therapeutic programs mice received normal or atherogenic diet for 28 weeks. Exercise training started after 14 weeks of dieting when atherosclerosis plaques were already established. Moderate intensity aerobic exercise training was performed on a motor treadmill for 60 min, 5 days/wk, during 14 weeks. Body mass, caudal blood pressure and heart rate were registered. Plasma lipoproteins were separated by FPLC and total cholesterol was determined by enzymatic methods. Cross sections of aortic root were stained with oil-red O for plaque size and fat content. Aorta longitudinal sections were stained with picro-sirius for collagen content. TNF- and IL-6 cytokines were measured by Elisa. Plasmatic MMP-9 was determined by zimography. Inflammatory tissue markers, MMP-9, CD40/CD40L and nitrotirosine, were measured by immunohistochemistry. We concluded that exercise training did not modify plaque size, but turned it into a more stable one by increasing its collagen content. Exercise training reduced plaque fat content, risk factors and plaque CD40 expression only in the preventive program. No difference in systemic inflammatory markers, and in plaque MMP-9 expression and nitrotirosine formation was noted.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Also available online. Includes bibliographical references (leaves 121-140).

Retinol (vitamin A) and its active metabolite, all-trans retinoic acid, signal through nuclear retinoic acid and retinoid X receptor (RAR/RXR) heterodimers. These complexes regulate the expression of genes involved in developmental processes such as limb development. In excess, retinoids are potent teratogens and cause marked reductive effects on the developing limb. The goal of this thesis was to elucidate the molecular mechanisms underlying retinoid-induced limb dysmorphogenesis. Specifically, using an in vitro limb culture system, I examined the involvement of one RAR isoform, RARgamma, in mediating retinoid insult. / My first objective was to examine how limbs deficient in RARgamma responded to exogenous retinoid exposure. I showed that RARgamma-null limbs (on an RARalpha1-null background) exhibited less severe limb defects following retinoid insult when compared to their wild-type counterparts. Additionally, the absence of RARgamma abolished the retinoid-induced misregulation of genes important for chondrogenesis (Sox9 and Col2a1 ) and limb outgrowth (Meis-1 and -2). / The next objective set out to determine how pharmacological activation of RARgamma affected limb development. The RARgamma-selective agonist (BMS-189961) caused limb dysmorphology (namely, effects on cartilage) that was comparable to pan-RAR activation with all-trans retinoic acid. A chondrogenesis-focused gene array analysis identified Mgp and Gdf10 as two RARgamma-responsive genes that may mediate retinoid-induced limb insult. / Subsequently, I assessed the functional involvement of Mgp in mediating retinoid teratogenicity. Limbs were treated with all- trans retinoic acid and warfarin (an inhibitor of MGP); warfarin co-treatment rescued limbs from retinoid-induced insult. / My final objective was to determine the importance of Gdf10 in mediating limb development. Recombinant human Gdf10-soaked beads were implanted into distal limb structures; ectopic overexpression of Gdf10 in the web (but not the digital ray) resulted in marked proximal limb malformations. / Collectively, these studies have illustrated the importance of RARgamma in retinoid teratology and have identified several potential mechanisms by which retinoids cause limb defects.

The development of T cells in the thymus is a tightly regulated process. Any defect in thymic differentiation could result in autoimmune disorders, inability to ward off infections or neoplasm. Early thymocyte development requires signals mediated through the preTCR complex by the associated CD3 chains (gamma, delta, epsilon, and zeta). Research conducted towards this project has revealed that signaling modules within the intracytoplasmic domain of CD3epsilon is absolutely required for this process. Interestingly, our results emphasized the importance of the proline-rich sequence motif in preTCR mediated signaling events, such as the proliferation of double negative thymocytes and the regulation of TCR surface expression on double positive thymocytes in a stage-specific manner. The outcomes of this project may provide a better understanding of the mechanism of preTCR-mediated thymocyte differentiation and the role of CD3 chains in these processes.

Distraction Osteogenesis (DO) is a technique for bone lengthening and filling of bone defects following trauma, infection or resection of tumors. DO consists of an osteotomy of the bone to be lengthened, followed by controlled distraction of the bone segments with an external fixator until the desired lengthening is obtained (distraction phase). This is followed by the consolidation phase, during which the external fixator is kept in place until the newly formed bone in the distracted zone consolidates. This phase is long and may cause numerous problems. Ongoing research aims at finding a method to accelerate the consolidation of the newly formed bone. / Fibroblast Growth Factors (FGF) play a significant role in bone development and repair. FGF 18 has been shown to be the only FGF member to be expressed throughout both the distraction and the consolidation phases of DO. It was also reported that FGF18 is the physiological ligand of FGFR3. Therefore, we hypothesized that FGF18 and FGFR3 may have an important role in DO. / To test this hypothesis, we investigated DO in FGFR3 deficient mice (FGFR3-/-). (FGF18 deficient mice are not viable). A miniaturized DO apparatus was applied to the tibia followed by an osteotomy. Distraction began after a 5-day latency period at a rate of 0.2 mm/12 hours for 12 days. / Samples were collected at 3 time points comparing the mutants (FGFR3-/-) to their wild type litter' sates: end of distraction (17 days post-surgery), mid-consolidation (34 days post-surgery), and end of consolidation (51 days post surgery). The samples were analyzed using X-ray, DEXA, microCT, histology, biomechanical testing and Real-Time PCR. / Our results revealed that FGFR3 deficient mice showed accelerated bone formation compared to the W.T. littermates at mid-consolidation where the parameters measured revealed increased bone mineral density, bone mineral content and trabecular number in the mutant tibial samples. The newly regenerated bone consolidated faster in the FGFR3 knock-out mice and the bone was of better quality as revealed by biomechanical tests in which more force was needed to break the mutant bone because it exhibited higher resistance than the age matched wild-type sample. The marker gene expression patterns revealed an up-regulation of chondrogenic markers that suggest that the knock-out mice follow the endochondral ossification pathway during DO. All results were statistically significant. / These results show that signaling through FGFR3 acts to decrease bone formation during DO. Consequently, blocking FGFR3 may lead to accelerated bone formation in DO. This may have important clinical implications in attempts to improve the functional outcome of DO by decreasing the long duration that the external fixator has to be kept on.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 255-274.