Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from thesis description page. Includes bibliographical references.

Thesis (Ph. D.)-- University of Washington, 2005. / Vita. Includes bibliographical references (leaves 126-142).

Chronic myeloid leukemia (CML) is characterized by clonal expansion of hematopoietic progenitor cells, result from the translocation (9:22). The oncogene BCR-ABL, in the Ph chromosome, is transcribed and translated into a fusion protein BCR / ABL. The ABL tyrosine kinase (TK) in the fusion protein is constitutively activated and is needed for the initial leukemogenic event of CML and its activity induces production of reactive oxygen species (ROS). Of particular relevance to CML is the fact that an increase of ROS can have consequences, facilitating genomic instability may contribute to disease progression. The aim of this study was to determine the oxidative status in patients with CML, in attendance at a university hospital (HUWC). This is a cross-sectional study consisted of 30 adult patients of both sexes with clinical and laboratory diagnosis of CML on treatment with inhibitors (TK) 1st and 2nd generation. The concentrations of malondialdehyde (MDA) and nitrite (NO2-) were performed by spectrophotometric method. The activities of enzymes glutathione peroxidase (GSH-Px) and catalase (CAT) were determined in hemolysate by Glutathione Peroxidase Cellular Activity Kit  Assay (Sigma-Aldrich) and spectrophotometry, respectively. Total glutathione, reduced glutathione (reduced GSH), glutathione (GSSG) were determined by Total Glutathione Activity Kit  (Assay Designs, Inc) and calculated the ratio GSH / GSSG. For statistical analysis of nonparametric data was used  and ANOVA test for multiple comparisons Tukey. It was considered the minimum level of significance of 5%. The average concentrations of NO2- and MDA were increased in CML patients compared to control, regardless of disease activity. The antioxidant profile was characterized by decreased CAT and GSH-Px increased also independent of disease activity. The reduced GSH is presented decreased, the GSSH, increased and GSH / GSSG decreased. It was observed that patients using protease inhibitors of TK 2nd generation of oxidative stress parameters were significantly elevated compared to controls. In the analysis of patients on imatinib were not detected significant changes in oxidative status. We conclude that patients with CML are under oxidative stress and impaired antioxidant activity. / A Leucemia mielÃide crÃnica (LMC) à caracterizada pela expansÃo clonal de cÃlulas progenitoras hematopoÃticas, resultante da translocaÃÃo (9:22). O oncogene de fusÃo BCR-ABL, no cromossomo Ph, à transcrito e traduzido numa proteÃna de fusÃo BCR/ABL. A tirosina quinase (TK) ABL na proteÃna de fusÃo à constitutivamente ativada sendo necessÃria para os eventos leucemogÃnicos iniciais da LMC e sua atividade induz a produÃÃo de espÃcies reativas de oxigÃnio (EROs). De particular relevÃncia para LMC à o fato de que um aumento de EROs pode ter consequÃncias, facilitando a instabilidade genÃmica podendo contribuir para a progressÃo da doenÃa. O objetivo do estudo foi determinar o perfil oxidativo em pacientes com LMC, em acompanhamento ambulatorial no Hospital UniversitÃrio Walter CantÃdio (HUWC). Trata-se de um estudo transversal constituÃdo de 30 pacientes adultos, com diagnostico clÃnico e laboratorial de LMC, em tratamento com inibidores de (TK) de 1 e 2 geraÃÃo. As concentraÃÃes de malonaldeÃdo (MDA) e de nitrito (NO2-) foram realizadas por mÃtodo espectofotomÃtrico. As atividades das enzimas Glutationa peroxidase (GSH-Px) e catalase (CAT) foram determinadas no hemolisado, por kit Glutathione Peroxidase Cellular Activity Assay (Sigma-Aldrich) e por espectofotometria, respectivamente. Glutationa total, glutationa reduzida (GSH reduzida), glutationa oxidada (GSSG) foram determinadas por kit Total Glutathione Activity (Assay Designs, Inc) e calculada a relaÃÃo GSH/GSSG. Para a anÃlise estatÃstica de dados nÃo paramÃtricos foi utilizado o ANOVA e o teste de mÃltiplas comparaÃÃes de Tukey. Foi considerado o nÃvel mÃnimo de significÃncia de 5%. As concentraÃÃes mÃdia de MDA e de NO2- foram aumentadas nos pacientes com LMC em relaÃÃo ao controle, independente da atividade da doenÃa. O perfil antioxidante foi caracterizado pela diminuiÃÃo da CAT e aumento da GSH-Px tambÃm independente da atividade da doenÃa. A GSH reduzida se apresentou diminuÃda, a GSSH, aumentada e a relaÃÃo GSH/GSSG diminuÃda. Os pacientes em uso de inibidores de TK de 2 geraÃÃo apresentaram parÃmetros do estresse oxidativo significativamente elevados em relaÃÃo ao grupo controle. Conclui-se que os pacientes com LMC estÃo sob estresse oxidativo e com atividade antioxidante comprometida.

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.

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The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic activity of SynPTP was examined toward several exogenous protein substrates that had been phosphorylated on either tyrosine residues or serine residues. SynPTP exhibited phosphatase activity toward tyrosine phosphorylated protein substrates (Vmax toward phosphotyrosyl 32P-casein was 1.5 nmol/min/mg). However, no phosphatase activity was detected toward serine phosphorylated protein substrates. SynPTP displayed phosphohydrolase activity toward several organophosphoesters including para-nitrophenyl phosphate (p-NPP), beta-napthyl phosphate and phosphotyrosine but not toward alpha-napthyl phosphate, phosphoserine, or phosphothreonine. Kinetic analysis indicated that the Km (0.6 mM) and Vmax (3.2 mmole/min/mg) values for SynPTP toward pNPP are similar to those of other known bacterial low molecular weight PTPs. The protein phosphatase activity of SynPTP was inhibited by sodium orthovanadate, a known inhibitor for tyrosine phosphatases, but not by okadaic acid, an inhibitor for many serine/threonine phosphatases. Mutagenic alteration of the predicted catalytic cysteine, Cys7, to serine abolished enzyme activity. Several phosphotyrosine containing proteins were detected from the whole cell extracts of Synechocystis sp. PCC 6803 through immunoreactions using anti-phosphotyrosine antibody. SynPTP was observed to dephosphorylate three of these proteins in vitro. Two of these proteins were identified by peptide-mass fingerprinting analysis, as PsaD (photosystem I subunit II) and CpcD (phycocyanin rod linker protein). In addition, several phosphotyrosine proteins were detected from the soluble and membrane fractions of Synechocystis sp. PCC 6803 cell extracts by in vitro substrate trapping as potential endogenous substrates of SynPTP. Two of these proteins were identified as the alpha and beta subunits of phycocyanin. We therefore speculate that SynPTP might be involved in the regulation of photosynthesis in Synechocystis sp. PCC 6803. / Ph. D.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 5 uppsatser.

The Cbl family of E3 ubiquitin ligases are important negative regulators of multiple receptor and cytoplasmic tyrosine kinases, and participate in a wide variety of cellular processes. Uncoupling of Cbl-mediated negative regulation allows activated receptor tyrosine kinases such as the Met receptor to escape degradation, enhancing their oncogenic potential in vitro and in vivo. Despite the consequences of loss of Cbl-mediated negative regulation for human disease, little is known about the mechanisms regulating Cbl protein levels themselves. / In this thesis work, I demonstrate a differential regulation of c-Cbl and Cbl-b downstream of the Met receptor tyrosine kinase. Cbl-b protein levels decrease in response to Met kinase activity, whereas c-Cbl levels remain stable. Cbl-b is partially degraded in a proteasome-dependant manner. This requires Cbl-b ubiquitin ligase activity and a carboxy terminal domain region located between the RING and UBA domains. I conclude that the regulation of c-Cbl and Cbl-b differs downstream of Met, and propose that negative regulation of Cbl-b by a dysregulated Met receptor may contribute to tumourigenesis.