No description available.

Insect haemolynph typically contains very high levels of free amino acids 50 1 00 times that which is normal for mammalian plasma. This study examines the modulatory effects of amino acids on fluid secretion and ion transport by isolated MTs of Rhodnius prolixus and Drosophila melanogaster. The results show that the secretion rates of isolated Malpighian tubules of both Rhodnius and Drosophila are modulated by the presence of specific amino acids in the bathing saline. Some amino acids are stimulatory, some are inhibitory and others have little or no effect. Glutamine appears to be particularly important as a stimulant of fluid secretion. As well, secreted fluid pH and Na +concentration increase and K+ concentration decreases in response to glutamine. Amino acids do not appear to be important as metabolite. in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Amino acids are known to be compatible osmolytes and may be acting to maintain cell homeostasis and thus to sustain fluid secretion. The passive movement of amino acids may result in cell volume changes, and some form of osmosensor is may be coupled to activation of specific kinases to produce the observed increases in fluid secretion. The effects of several kinase inhibitors were therefore examined. The glutamine dependent increase in MT fluid secretion is blocked by two inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumoral. Inhibitors of other kinases (PKA, PKC, PKG, PI-3, p38, ERK and MEK), did not block glutamine's effects on fluid secretion rate. Alterations in cytoskeletal structure appear not to be required because cytoskeletal disrupting agents did not block the glutamine dependent inc~ease in fluid secretion, nor was the increase dependent upon protein synthesis. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect Malpighian tubules. / Thesis / Master of Science (MS)

INTRODUÇÃO: O melanoma primário da cavidade oral é uma neoplasia agressiva, rara e originada a partir da proliferação de melanócitos malignos da mucosa. Ele representa aproximadamente de 0,2 a 8% de todos os melanomas. Estudos recentes apontam algumas vias moleculares tem sido encontradas por estarem envolvidas na patogenia dos melanomas. Dentre essas vias destaca-se a via proliferativa da MAPK (mitogen activated protein kinase), esta cascata de sinalização está envolvida no controle do crescimento celular, proliferação e migração, e tem sido relacionada com um papel importante no desenvolvimento e progressão do melanoma cutâneo. OBJETIVOS: Analisar a expressão proteica e mutação pontual dos componentes da via MAPK e correlacionar com os dados clínicos-histológicos. MATERIAL E MÉTODOS: Através da imunoistoquímica avaliar a expressão proteica dos anticorpos RAS; BRAF; MEK1; MEK2; ERK1 e ERK2 em 35 casos de melanomas orais organizados em matriz (TMA: Tissue Microarray) e através de pirosequenciamento avaliar a mutação pontual dos genes BRAF; NRAS; KRAS em 14 casos de melanomas orais. RESULTADOS: Idade dos pacientes entre 9 e 91 anos, sem predileção por sexo, 75% caucasianos, 71,42% acometeram o palato, 80% com aspecto histológico grau III. A análise da expressão proteica foi: RAS (28,57%); BRAF (82,85%); MEK1 (0%); MEK2 (51,43%); ERK1 (20%)e ERK2 (74,28%). Na análise molecular observamos mutações para BRAF (9/14 casos) e NRAS (2/14 casos). CONCLUSÃO: Todos os aspectos da via MAPK necessita de outras elucidações em melanomas de áreas foto-protegidas e melanomas de mucosa e comparando diferentes populações. Entretanto, os resultados deste presente estudo apontam importante alterações na cascata RAS-RAF-MEK-ERK e estes são indicadores de prognóstico ruim em melanomas primários da mucosa oral, independente da exposição solar / BACKGROUND: Primary melanoma of the oral cavity is an aggressive and rare neoplasm and originated from the proliferation of malignant melanocytes of the mucosa. It represents approximately 0.2 to 8% of all melanomas. Recent studies indicate some molecular pathways have been found to be involved in the pathogenesis of melanomas. Among these means there is a proliferative MAPK pathway (\"mitogen activated protein kinase\"), this signaling pathway is involved in controlling cell growth, proliferation and migration, and it has been associated with a role in the development and progression of melanoma skin. OBJECTIVES: To analyze protein expression and mutation of components of the MAPK pathway and to correlate with the clinical, histological data. MATERIALS AND METHODS: Using immunohistochemistry to evaluate the protein expression of RAS, BRAF, MEK1, MEK2, ERK1 and ERK2 antibodies in 35 cases of oral melanomas organized array (TMA: Tissue Microarray) and using pyrosequencing to assess the mutation of the BRAF, NRAS, KRAS in 14 cases of oral melanomas. RESULTS: Age of patients between 9 and 91 years, regardless of gender, 75% Caucasian, 71.42% in palate, 80% with histologic grade III. Analysis of protein expression was: RAS (28.57%); BRAF (82.85%); MEK1 (0%), MEK2 (51.43%); ERK1 (20%) and ERK2 (74.28%). Molecular analysis we found BRAF mutations (9/14 cases) and NRAS (2/14 cases). CONCLUSION: All aspects of the MAPK pathway requires further elucidation in melanomas of photo-protected areas and mucosal melanomas and comparing different populations. However, the results of this study indicate important changes in the cascade RAS-RAF-MEK-ERK and these are indicators of poor prognosis in primary melanomas of the oral mucosa, regardless of sun exposure

Ip Wai-Ki. / "July 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 261-290). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH. In the myocardium, NHE1 has been implicated in ischemia/reperfusion (I/R) and cardiac hypertrophy (CH). Hormonal, autocrine and paracrine stimuli, acidosis, cardiotoxic metabolites released during I/R and CH increases NHE1 protein expression and activity. The involvement of NHE1 in CH and I/R has been further supported with the use of NHE1 inhibitors, which have been beneficial in the prevention/regression of several models of CH and I/R injury. Despite the fact that elevation of NHE1 expression and activity have been demonstrated in several models of heart disease, it was unclear whether elevation of NHE1 protein expression was sufficient to induce a specific cardiac pathology, or whether activation of the protein was required. To understand the direct role of NHE1 in CH and I/R, an in vivo and in vitro gain-of-function model, expressing varying levels and activities of NHE1 were examined. In vivo, our N-line mice expressed wild type NHE1 and our K-line mice expressed constitutively active NHE1. In vitro, neonatal rat ventricular cardiomyocytes were infected with the IRM adenovirus containing wild type NHE1 or the K-IRM adenovirus containing active NHE1. We demonstrated that expression of constitutively active NHE1 promotes CH to a much greater degree than expression of wild type NHE1 alone, both in vivo and in vitro. This NHE1-dependent hypertrophic response occurred independent of signaling pathways involved in CH including, mitogen activated protein kinases, p90 ribosomal S6 kinase, calcineurin and glycogen synthase kinase. The NHE1-dependent hypertrophic effect also occurred independent of gender. In addition, the expression of active NHE1 increased the susceptibility of intact mice to neurohormonal stimulation and progressed the hypertrophic response. When these hearts expressing active NHE1 were subjected to I/R using the ex vivo working heart perfusion model, fatty acid (FA) oxidation and glycolysis rates increased, thus generating greater ATP production rates. This was associated with cardioprotective effects in the myocardium, as well as a more energetically efficient myocardium. Expression of the endoplasmic reticulum (ER) stress response proteins, calreticulin and PDI were also shown to be increased relative to controls, and may contribute to the cardioprotection observed. We demonstrate that active NHE1 induces cardioprotection and alters cardiac metabolism in working hearts subjected to I/R. Overall, our results suggest that expression of active NHE1 has a double edged sword effect, on one side it induces CH while on the other side, it protects the heart against I/R injury.

No description available.

No description available.

L'étirement est un stimulus hypertrophique qui active de nombreuses voies de signalisation similaires à celles mises en évidence lors de l'étude de l'hypertrophie cellulaire. L'objectif principal de mon travail de thèse était de caractériser les évènements moléculaires impliqués dans l'activation des MAPKinases (MAPK), ERK et JNK lors de l'étirement. Nous avons étudié ces protéines par 2 approches différentes. D'une part, nous nous sommes intéressés aux rôles de protéines potentiellement nécessaires à l'activation des MAPK. D'autre part, nous avons cherché à mettre en évidence des interconnexions moléculaires entre les différentes voies de signalisation activées par l'étirement cellulaire, en montrant notamment la formation de complexes protéiques nécessaires à l'activation des différents partenaires. Nous montrons ainsi que deux protéines à activité tyrosine kinase, l'Epidermal Growth Factor Receptor (EGFR) et la Proline-rich tyrosine kinase 2 (Pyk2), sont respectivement nécessaires à l'activation de ERK et de JNK lors de l'étirement. Ces cascades de transduction peuvent être dépendantes de la petite protéine G Ras. Bien que les voies des MAPK et de PI3K/Akt soient considérées comme indépendantes, nous montrons également que Akt participe à l'activation de ERK par l'étirement. Enfin, nous avons montré la formation d'un complexe Protein Kinase C (PKC)/Calcineurine nécessaire à l'activation et à la translocation de la PKC lors de l'étirement. Cette étude de différentes voies de signalisation et des interactions protéiques apporte une meilleure connaissance des mécanismes activés par l'étirement cellulaire et permet donc de mieux comprendre la signalisation impliquée dans l'hypertrophie ventriculaire

[Truncated abstract] The pro-inflammatory cytokine tumour necrosis factor (TNF) has a critical role in skeletal muscle atrophy. The catabolic effect of TNF is partially due to abrogation of the anabolic insulin-like growth factor 1 (IGF-1) signalling pathway. However, the precise signalling events that lead to the loss of myofibrillar protein following activation of TNF receptor are unknown. The over arching aim of the study is to determine the mechanisms of by which TNF induces atrophy in differentiated muscles cells. To achieve this aim a series of experiments were performed to: 1) investigate the molecular events that lead to TNF mediated myofibre atrophy, 2) determine to what extent c-Jun N-terminal Kinase (JNK) signalling plays a part in TNF induced myotube atrophy, and in TNF-mediated inhibition of IGF-1 induced hypertrophy, and 3) use inhibitors of JNK to block the catabolic effects of TNF. 1) To investigate the molecular events that lead to TNF mediated myofibre atrophy, the experiments were conducted using C2C12 mouse myotube cultures and primary myotube cultures derived from FVB mice, and transgenic mice which over-express Class 2 IGF-1 Ea in skeletal muscles (IGF:C2). The treatment of mature C2C12 and FVB primary myotubes (respectively at 7 and 4 days after fusion medium) with 10 ng/mL of TNF for 3 days resulted in statistically significant myotube atrophy (decreased mean width). The observed TNF-mediated atrophy has not previously been demonstrated in tissue cultured myotubes. In contrast, addition of IGF-1 (20 ng/ml) to 7 day C2C12 myotubes for 3 days resulted in significant hypertrophy. ... The most suitable inhibitor was TAT-TIJIP and was thus used in subsequent studies. Inhibition of JNK activity by TAT-TIJIP was confirmed indirectly by detecting nuclear translocation of c- Jun, which is a downstream target of phosphorylated JNK. Immunohistochemical analyses showed nuclear localisation and phosphorylation of c-Jun in TNF treated myotubes. Nuclear localisation and phosphorylation of c-Jun was not observed in cultures pre-treated with TAT-TIJIP before TNF treatment, nor in the untreated control myotubes. 3) The use of JNK inhibitors to block the catabolic effects of TNF was tested using C2C12 and primary myotube cultures. Pre-treatment of C2C12 and primary FVB myotubes with the JNK inhibitor TAT-TIJIP, 30 min before TNF administration (for 3 days) prevented myotube atrophy. The mean width of myotubes pre-treated with TATTIJIP prior to TNF treatment closely resembled that of the control myotubes. Administration of TNF in combination with TAT-TIJIP for 3 days to C2C12 myotubes prevented myotube atrophy and unexpectedly resulted in hypertrophy when compared to the mean widths of untreated and TAT-TIJIP treated myotubes. This trend was also demonstrated in the FVB primary cultures. These combined results strongly support the role of JNK in TNF-mediated atrophy. Preliminary studies were carried out in vivo using the mdx mouse model of muscular dystrophy, TAT-TIJIP was administered via intraperitoneal injection to the mice for 3 days at a dose of 10 mg/ml, however the results form this study are inconclusive. These novel observations are of considerable interest to the field of muscle wasting because they demonstrate for the first time TNF-mediated myotube atrophy, the role of JNK in situations of TNF induced muscle atrophy, and explore the use of JNK inhibitors to prevent muscle atrophy.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.