Decreased respiratory muscle strength and/or excessive loads imposed on the respiratory muscles by disease may result in respiratory muscle fatigue and ventilatory failure. Once the respiratory muscles fatigue, the only treatment is rest by mechanical ventilation. However, no one has yet determined the best protocol of rest. The purpose of these studies was to develop an animal model in the hamster in order to examine the time course of recovery following fatigue of the diaphragm and specifically, to test whether mechanical ventilation or spontaneous unloaded breathing was a better mode for functional recovery. The studies required the initial development of an anesthetic regimen which produced minimal respiratory depression in the hamster. A new method of stimulating the diaphragm in small animals was developed by apposing plate electrodes directly against the diaphragm. The validity of this technique was examined and comparison of the mechanical and electrophysiological response to that of phrenic nerve stimulation were similar at maximal stimulation. The histological characteristics of the normal hamster diaphragm were determined for fibre type proportions and sizes, oxidative capacity and glycogen levels in the costal and crural regions of this muscle. The examination revealed three distinct areas of the diaphragm with different histological features: the abdominal surface of the crural region, the thoracic surface of the crural region and the sternal and costal region. Diaphragmatic fatigue was induced in vivo by repetitive electrical stimulation which resulted in both high and low frequency fatigue. The fatigue stimulus also produced muscle fibre damage, primarily along the abdominal surface of the diaphragm over the electrodes, and glycogen depletion in the type lib fibres. Rest by continuous mechanical ventilation resulted in recovery of high frequency fatigue in the hamster diaphragm whereas rest by spontaneous unloaded breathing resulted in no recovery. Sham fatigue groups rested by either mechanical ventilation or spontaneous breathing demonstrated progressive deterioration in transdiaphragmatic pressure throughout the rest period. Decreased muscle fibre damage but increased inflammation and glycogen depletion was demonstrated in all four fatigue/sham fatigue and rest groups compared to that demonstrated by the fatigue/sham fatigue only groups. The results suggest that passive rest by continuous mechanical ventilation promotes recovery following fatigue induced by electrical stimulation. Additional factors such as prolonged fasting, loads imposed on the diaphragm by the plate electrode apparatus, positive pressure ventilation, and cumulative effects of intraperitoneal urethane likely contributed to the progressive deterioration of diaphragmatic function demonstrated in the animals of the two sham groups rested by either spontaneous breathing or mechanical ventilation, and confounded the results shown by the two fatigue groups rested by either spontaneous breathing or mechanical ventilation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Proefschrift--Groningen.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-05-31T19:55:30Z No. of bitstreams: 1 Adenizar Chagas Júnior. O Imprint como método para detecção de Leptospira SSP. 2009.pdf: 396164 bytes, checksum: 8f423288d4f795e861df22e9de3acb59 (MD5) / Made available in DSpace on 2012-05-31T19:55:30Z (GMT). No. of bitstreams: 1 Adenizar Chagas Júnior. O Imprint como método para detecção de Leptospira SSP. 2009.pdf: 396164 bytes, checksum: 8f423288d4f795e861df22e9de3acb59 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Na determinação da eficácia de novas candidatas à vacina para leptospirose, o marcador primário considerado é a mortalidade, e um marcador secundário importante é a indução de uma imunidade estéril. Entretanto, a avaliação da imunidade estéril é dificultada pelo tempo demandado e pela complexidade de métodos como o isolamento pela cultura. Neste estudo, foi avaliado o uso do método do imprint (ou touch preparation) na detecção da presença de leptospiras em tecidos de hamsters infectados com L. interrogans sorovar Copenhageni. Comparado com a cultura, o imprint demonstrou igual ou melhor detecção de leptospiras em amostras de rim, fígado, pulmão e sangue coletadas após a infecção obtendo uma concordância geral boa (κ = 0.61). Além disso, na avaliação de hamsters imunizados com uma proteína recombinante de Leptospira candidata à vacina e subsequente desafio com leptospiras patogênicas, a concordância entre a cultura e o imprint foi alta (κ = 0.84). Estes achados indicam que o imprint é um método rápido para a observação direta de Leptospira spp. e que pode ser facilmente aplicado na avaliação de animais infectados experimentalmente com leptospiras e na determinação de imunidade esterilizante durante avaliações de potenciais candidatas à vacina. / In determining the efficacy of new vaccine candidates for leptospirosis the primary endpoint is death and an important secondary endpoint is sterilizing immunity. However, evaluation of this endpoint is often hampered by the time consuming demands and complexity of methods such as culture isolation (CI). In this study, we evaluated the use of an imprint (or touch preparation) method (IM) in detecting the presence of leptospires in tissues of hamsters infected with L. interrogans serovar Copenhageni. Compared to CI, the IM exhibited equal or improved detection of leptospires in kidney, liver, lung and blood samples collected post-infection and the overall concordance was good (κ = 0.61). Furthermore, in an evaluation of hamsters immunized with a recombinant Leptospira protein-based vaccine candidate and subsequently challenged with leptospires, the agreement between the CI and IM was very good (κ = 0.84). These finding indicate that the IM is a rapid method for the direct observation of Leptospira spp. that can be readily applied to evaluating Leptospira infection in experimental animals and determining sterilizing immunity when screening potential vaccine candidates.

The last step in sphingomyelin's de novo biosyn-thetic pathway was investigated in Baby Hamster Kidney (BHK-21F) cells in tissue culture. Three types of pulse-chase experiments were done to try to identify the precursor for sphingomyelin's phosphocholine moiety. First, [Me- ³H]-choline was used to monitor the movement of the choline moiety in all the possible phosphocholine donors: ie. phosphocholine, CDP-choline, phosphatidylcholine, lysophospha-tidylcholine, and glycerophosphocholine. Radioactivity was observed in phosphocholine before appearing in phosphatidylcholine, glycerophosphocholine, and sphingomyelin. Specific radioactivities of phosphatidylcholine and sphingomyelin revealed a peculiar pattern, if representative of a precursor-product relationship between these two phospholipids. Their specific radioactivities became equal at 22 hours of chase and remained quite similar for the next 24 hours. The other two types of pulse-chase experiments both utilized prelabeled BHK phosphatidyl[Me- ³H]choline in phospholipid vesicles as their 'pulse' source. Phospholipid Exchange Protein(PLEP)-mediated exchange and polyethylene glycol/phytohemagglutinin (PEG/PHA)-mediated fusion between phospholipid vesicles and BHK cells were used to introduce the labeled phosphatidylcholine. In addition, in the 'fusion' experiments, other labeled compounds (glycerophosphocholine and sphingomyelin) were substituted for labeled phosphatidylcholine. PLEP- mediated exchange of labeled phosphatidylcholine did not result in enough transfer of radioactivity into the cell to adequately monitor individual cell phospholipids or any transfer of label - ie. from phosphatidylcholine to sphingomyelin. However, PEG/PHA-mediated fusion of vesicles and cells did result in enough radioactivity showing up in the cells. When labeled phosphatidylcholine was used, the radioactivity ratio between it and sphingomyelin averaged around 16, depending on the length of the chase (2-52 hours)m The use of either labeled glycerophosphocholine or sphingomyelin resulted in a ratio of about 1.8. One 'cold-trap1 experiment was done by including a large amount of unlabeled glycerophosphocholine with labeled phosphatidylcholine in the vesicle preparation. The resultant radioactivity in sphingomyelin was 50% less than previously, but phosphatidylcholine's had remained the same. The evidence seems to indicate a reversible precursor-product relationship between phosphatidylcholine and sphingomyelin, but does not clearly show whether or not any other intermediate (such as glycerophosphocholine) is also involved. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Thesis (Ph.D.)--University of Wisconsin--Madison, 2007. / Includes bibliographical references. Also available on the Internet.

Thesis (M.Sc. (Anatomy))--Mahidol University, 1983.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2007. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The tenuissimus is a long, thin hindlimb skeletal muscle which in hamsters contains about 200 extrafusal muscle fibers. Embedded in this richly innervated muscle is a continuous array of 16-20 closely packed muscle spindles suggesting that it may play a role in hindlimb proprioception. This high spindle density also makes the muscle ideal for the isolation and harvesting of these sensory receptors. In this correlative light and electron microscopic study, freshly frozen specimens were first prepared for serial microscopic analysis. Camera lucida reconstruction of spindle distribution showed a close proximity to the main artery and nerve in the central core of the muscle. Oxidative enzyme and myosin ATPase staining profiles were examined in both the intrafusal and extrafusal fiber populations. Type I and type II extrafusal fibers were present in even numbers and were distributed evenly throughout muscle cross-sections. Enzyme staining varied along the lengths of the three intrafusal fiber types. The fine structure of spindles was examined using transmission (TEM), conventional scanning (SEM), and high resolution scanning electron microscopy (HRSEM). For conventional SEM, isolated spindles were first fixed in 2.5% buffered glutaraldehyde, followed by 1% osmication, and mechanical disruption of the outer capsule under the dissecting microscope. Preparation for HRSEM included aldehyde/osmium fixation and freeze-cleavage of entire tenuissimus muscles in liquid N₂. Selective extraction of the cytosol with 0.1% OsO4 permitted the visualization of numerous intracellular structures. In these specimens, the capsular sleeve showed a multilayered pattern of vesicle-laden cells with variant surface topography in certain locations. Punctate sensory nerve endings adhered intimately to the surfaces of underlying intrafusal fibers in the equatorial and juxtaequatorial regions. By TEM and HRSEM these endings appeared crescent-shaped and were enveloped by external laminae. Each profile contained a plethora of mitochondria and cytoskeletal organelles. The methodology used in this study provides, for the first time, a three-dimensional view of the exquisite cytological architecture of this neuromuscular receptor. / Medicine, Faculty of / Graduate

A patogenia na leishmaniose visceral (LV) não é totalmente conhecida. Em LV em hamsteres avaliamos a participação de imunoglobulinas nas lesões. IgG foi detectada no sinusóide hepático, nos septos pulmonares e nos capilares glomerulares, em maior intensidade aos 30 e 45 dias pós-infecção (PI); C3 estava ausente, exceto no rim. Os dados não sendo compatíveis com a deposição de imunocomplexo, estudamos ultraestruturalmente a internalização de imunoglobulinas. No fígado e no rim a quantidade de imunoglobulina em célula endotelial era maior aos 30 dias PI em relação ao controle não infectado e no pulmão, aos 30 dias PI em relação aos 60 dias PI. Imunoglobulina internalizada por célula endotelial observada neste estudo na LV em hamsteres, marcadamente aos 30 dias PI, pode ser um mecanismo alternativo de lesão na LV / Pathogenesis of visceral leishmaniasis (VL) is not fully known. In VL in hamsters we evaluated the participation of immunoglobulins in the lesions. IgG was detected in the hepatic sinusoid, in the lung alveolar walls and in the glomerular capillaries in higher intensity at 30 and 45 days post-infection (PI); there were no C3, except in the kidney. Since the data were not compatible with immune complex deposition, we studied ultraestructurally the internalization of immunoglobulins. In the liver and in kidney the amount of immunoglobulin within endothelial cells was greater at 30 days PI than in non infected control, and in the lung at 30 days PI in relation to 60 days PI. Immunoglobulin internalized by endothelial cells observed in VL in hamsters, remarkably at 30 days PI, may be an alternative mechanism of lesion in VL

Made available in DSpace on 2015-11-11T12:13:57Z (GMT). No. of bitstreams: 2 elvia_esparza_ioc_dout_2014.pdf: 5839066 bytes, checksum: 29e89c3117f3207c7a4df861cc312204 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2015-06-10 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A expressão de arginase-1 (Arg1) está associada com a patogênese da leishmaniose visceral (LV), embora os mecanismos que norteiam a sua regulação na doença não sejam adequadamente conhecidos. Nesta tese foram explorados os mecanismos de regulação da Arg-1 no modelo experimental hamster, a fim de aplicar o conhecimento para o tratamento da doença. Encontrou-se que células infectadas in vitro com Leishmania (Leishmania) donovani expressaram Arg-1 e inhibiram a produção do óxido nítrico, o que contribuiu para o aumento da carga parasitária. A regulação da Arg1 dependeu do fator de transcrição STAT-6, que foi ativado de forma independente da IL-4. Para explorar outros mecanismos envolvidos na ativação de STAT-6 foi utilizada uma biblioteca quimica de 80 inibidores dos receptores tirosine quinases (RTK) e uma membrana microarranjo revestidas com anticorpos contra proteínas phosphoriladas dos RTK. Desta forma, foi possível identificar a ativação do receptor do fator de crescimento de fibroblasto-1 (FGFR-1) e o receptor do fator de crescimento semelhante à insulina tipo I (IGF-IR) nos macrofagos. Além disso, realizou-se uma caracterização mais detalhada da cascata de fosporilação canônica dessos receptores. Experimentos in vitro mostraram que macrófagos infectados com L. donovani aumentaram a transcrição de Arg1 quando foram tratados com os ligantes dos RTK, o FGF-2 e o fator de crescimento semelhante à insulina tipo 1 (IGF-I), sugerindo que estes RTK tinham um papel na regulação da Arg1 na LV Quando inibidores químicos do FGFR-1 e IGF-IR foram testados em macrófagos infectados, a Arg1 e o STAT-6 foram inibidos sugerindo uma ligação entre essas vias. De fato, a estimulação com FGF-2 e IGF-I aumentou a fosforilação e a ativação transcripcional do STAT-6, e se observou um efeito aditivo quando a citocina IL-4 foi adicionada, demonstrando a interação positiva destas vias. A inibição do STAT-6 mediante interferência de RNA impediu ou diminuiu significativamente a Arg-1 induzida nos macrófagos infectados e estimulados com FGF-2 e por IGF-I. respectivamente, indicando um papel chave do STAT-6 na Arg-1 induzida pelos RTK. A relevância destes achados experimentais para os seres humanos foi avaliada em cultivos de sangue total de doadores sadios estimulados in vitro e no plasma de crianças durante a doença ativa, um mês, seis meses e até um ano após tratamento. Foi observado um aumento de Arg-1 após o estimulo in vitro com IL-4 e FGF-2 ou IGF-I, consistente com o achado no modelo hamster. Nos pacientes, os níveis plasmáticos de Arg-1 não mudaram com a evolução da doença após e o tratamento e o FGF-2 só apresentou um aumento associado com a resolução um ano após tratamento. Em conclusão, os sinais comuns ativados através do FGF-1R e IGF-IR convergem com a IL-4 para ativar STAT-6 e a transcrição de Arg-1 na LV experimental, mas determinar o papel destes fatores de crescimento na LV humana requer estudos adicionais. Este estudo propõe a base mecanicista da regulação da Arg1 mediada pela convergência da sinalização RTK e IL-4R ao STAT-6 na LV e sugere que os inibidores RTK são de utilidade potencial como tratamento adjuvante da doença / Arginase - 1 (Arg1) expression is associated with the pathogenesis of visceral leishmaniasis (VL) but the mechanisms that lead to its up - regulation on disease are not completelly k nown . The objective of this thesis project was to explore the mecanistic ba sis of Arg - 1 regulation in the experimental hamster model of VL with the aim to apply the knowledge to the treatment of the disease. We found that cells infected in vitro with Leishmania donovani expressed Arg1 and that nitric oxide production was inhibite d , contibuting to the increased parasite burden. Arg - 1 regulation was dependent of the transcription factor STAT - 6, but the activation of this transcription factor was independent of IL - 4 cytokine. To explore other mechanisms involved in activation of STAT - 6 and the expression of Arg1 we used a chemical library of 80 tyrosine kinase receptor (RTK) inhibitors and a protein array with antibodies against phosphorylated RTKs. This allowed identify the activation of fibroblast growth factor receptor - 1 (FGFR - 1) a nd insulin - like growth factor receptor (IGF - IR) and the canonical down - stream signaling cascade. Macrophages infected in vitro with L. donovani increased transcription of Arg1 when treated with RTK ligands (FGF - 2, IGF - I), suggesting a role of these RTKs in the regulation of the Arg1 in LV. C hemical inhibitors of the activated receptors were used to assess the effect on infected macrophages. We found that inhibitors of FGFR - 1 and IGF - IR inhibited parasite burden, Arg1 and STAT - 6, suggesting a connection betw een these routes. Furthermore, the simultaneous stimulation of RTKs with its ligands FGF - 2 or IGF - I and IL - 4 increased the phosphorylation and transcriptonal activation of STAT - 6 and increased Arg1 in an additive manner, showing a positive interaction betw een these signaling cascades. STAT - 6 inhibition by RNA interference prevented or significantly diminished the induction of Arg - 1 in macrophages stimulated with FGF - 2 and IGF - I respectively, indicating a key role of the STAT - 6 on Arg - 1 induced by RTKs. The relevance of these experimental findings to humans was evaluated in stimulated whole blood cultures of healthy donor’s o r plasma of children during active disease, 1 month, 6 months or after 1 year of treatment . We found that the Arg - 1 induced by in vitro stimulation with IL - 4 and FGF - 2 or IGF - I, was consistent with the animal model. However, in the patients plasmatic Arg - 1 did not change over the follow - up, while FGF - 2 increased only after 1 year of treatment. We conclude that the signals activated throu gh FGFR - 1 and IGF - I converge to IL - 4 to activate STAT - 6 and induce Arg - 1 in experimental VL . This study propose that the mecanistic basis of the regulation of Arg1 in VL is mediated by the convergence of RTK signaling pathways and IL - 4R to STAT - 6 and sugge st the potential utility of RTK inhibitors as adjuvant treatment for the disease.