Growth and proliferation are normal functions of cells mediated in part via receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor (EGFR). The EGFR binds the extracellular signaling ligand EGF and transduces the signal into the cell. Phosphatidylinositol 3'-kinase (PI3K) responds to EGFR activation and initiates downstream signaling cascades responsible for cell cycle entry, proliferation and inhibition of apoptosis. Cell cycle arrest is required to stop cell growth and proliferation as well as allow apoptosis, if required. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) directly opposes PI3K signaling since its substrate is the PI3K product phosphatidylinositol 3,4,5-trisphosphate. PI3K is a heterodimer composed of a p85 regulatory subunit and a p110 catalytic subunit. PTEN is an essential tumor suppressor protein. Absence of PTEN has been associated with several types of cancer. <p>Our laboratory has characterized new specialized functions for the p85 protein. One function discovered was the ability of p85 to enhance PTEN lipid phosphatase activity. In this thesis PTEN activity is shown to be enhanced at least 3.5 fold in vitro by an equimolar amount of p85. <p>We performed an analysis of PTEN using seven PTEN mutants. Two types of mutants were created: i) regulatory or possible regulatory phosphorylation sites were substituted to mimic both phosphorylated and non-phosphorylated states and ii) alanine substitution of basic amino acid residues. The phosphorylation sites altered were the casein kinase 2 phosphorylation sites in the regulatory domain and tyrosine 336, a proposed regulatory phosphorylation site. Three mutants involving alanine substitution for basic amino acid residues included one mutant in the PASE domain and two more mutants in the C2 domain. It was observed that GFP-PTEN translocates to the plasma membrane upon EGF stimulation. The mimic of constitutive phosphorylation of the Casein kinase 2 sites resulted in cytoplasmic localization whereas the non-phosphorylated mimic was plasma membrane localized regardless of EGFR activation status. Neutralization of positive charge in the PASE and C2 domains seriously impeded the ability of PTEN to bind to phosphorylated phosphatidylinositol lipids and abolished the ability of the protein to translocate to the plasma membrane in response to receptor activation. Located within a cluster of positively charged lysine residues in the C2 domain is a potential phosphorylation site at tyrosine 336. The phosphorylation mimic showed decreased binding to some membrane lipids compared to the non-phosphorylated mimic. The results we generated are consistent with a current model for PTEN regulation that proposes PTEN is localized to the cytoplasm in quiescent cells and dephosphorylation of the regulatory domain occurs upon EGF stimulation allowing translocation to the plasma membrane. The model proposes that dephosphorylation of the casein kinase 2 sites unmasks regions of positive charge that interact with the anionic plasma membrane. Furthermore, the results suggested that at the plasma membrane p85 interacts with PTEN to increase lipid phosphatase activity and may be involved in targeting PTEN to the activated receptor where PI3,4,5P3 lipids are being produced.

Protein phosphorylation is a prevalent reversible post-translational modification that influences protein functions. The advent of phospho-proteomic technologies now enables proteome-wide quantitative detection of residues phosphorylated under different physiological conditions. The functional consequences of the majority of these phosphorylation events are unknown. This calls for endeavors to characterize their molecular functions and cellular effects. This can be facilitated by systematic approaches to categorize phosphorylation events, interpret their importance and infer their functions. I carried out comparative, evolutionary and integrative analyses on in vivo phosphorylation events to address these challenges. First, I performed cross-species comparative phospho-proteomic analysis to identify evolutionarily conserved phosphorylation events in human. A sequence alignment approach was used to identify phosphorylation events conserved at similar sequence positions across orthologous proteins and a network alignment approach was applied to identify potential evolutionarily conserved kinase-substrate interactions. Conserved human phosphoproteins identified are found enriched for proteins encoded by known cancer- and disease-associated genes. Next, I developed a new approach to analyze the sequence conservation of known phosphorylated residues on human, mouse and yeast proteins that factored in the background mutational rates of protein and phosphorylatable residue. Furthermore, sites were analyzed according to (i) characterized functions, (ii) prevalence, (iii) stoichiometry, their occurrence in (iv) structurally disordered/ordered protein regions, in (v) proteins of various abundance and in (vi) proteins with different protein interaction propensity to identify the factors influencing sequence conservation of phosphorylated residues. Importantly, my analysis suggests that false positives and randomly phosphorylated residues are present in existing phosphorylation datasets and they are more common on high abundance proteins. Lastly, I characterized the theoretical maximum phosphorylation capacity in terms of phosphorylatable residues and discovered that genomic tyrosine frequency correlates negatively and significantly with tyrosine kinase frequency and cell type in metazoan. This observation suggests that fidelity of phosphotyrosine signaling occurred partially through global tyrosine depletion.

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

Protein phosphorylation is a prevalent reversible post-translational modification that influences protein functions. The advent of phospho-proteomic technologies now enables proteome-wide quantitative detection of residues phosphorylated under different physiological conditions. The functional consequences of the majority of these phosphorylation events are unknown. This calls for endeavors to characterize their molecular functions and cellular effects. This can be facilitated by systematic approaches to categorize phosphorylation events, interpret their importance and infer their functions. I carried out comparative, evolutionary and integrative analyses on in vivo phosphorylation events to address these challenges. First, I performed cross-species comparative phospho-proteomic analysis to identify evolutionarily conserved phosphorylation events in human. A sequence alignment approach was used to identify phosphorylation events conserved at similar sequence positions across orthologous proteins and a network alignment approach was applied to identify potential evolutionarily conserved kinase-substrate interactions. Conserved human phosphoproteins identified are found enriched for proteins encoded by known cancer- and disease-associated genes. Next, I developed a new approach to analyze the sequence conservation of known phosphorylated residues on human, mouse and yeast proteins that factored in the background mutational rates of protein and phosphorylatable residue. Furthermore, sites were analyzed according to (i) characterized functions, (ii) prevalence, (iii) stoichiometry, their occurrence in (iv) structurally disordered/ordered protein regions, in (v) proteins of various abundance and in (vi) proteins with different protein interaction propensity to identify the factors influencing sequence conservation of phosphorylated residues. Importantly, my analysis suggests that false positives and randomly phosphorylated residues are present in existing phosphorylation datasets and they are more common on high abundance proteins. Lastly, I characterized the theoretical maximum phosphorylation capacity in terms of phosphorylatable residues and discovered that genomic tyrosine frequency correlates negatively and significantly with tyrosine kinase frequency and cell type in metazoan. This observation suggests that fidelity of phosphotyrosine signaling occurred partially through global tyrosine depletion.

The present study exploits two different molecules as biorecognition elements for biosensing. In the first case, a protein biosensor was performed using maltose-binding protein (MBP). The ability to manipulate protein function rationally also offers the possibility of creating new proteins of biotechnological value. Our design has been used to test the understanding of allosteric transitions in proteins. Here we examined a simple conformational change that can represent the biorecognition principle for a reagentless biosensor. Previously, modular strategies for transducing ligand-binding events into fluorescent and electrochemical responses have been reported. Starting with a study of the conformational changes of MBP this research will further develop electrochemical maltose biosensors. The responses of four individual mutations (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; and K25C-MBP-MT) were evaluated using square wave voltammetry. The possibility of using this type of transduction mechanism for sensor configurations and analyte specificity is discussed.The second part of this work involves SELEX (systematic evolution of ligands by exponential enrichment) and aptamers as biorecognition molecules. As a result of the SELEX method, we can obtain oligonucleotide sequences (aptamers) with recognition properties similar to antibodies. These synthetic elements play an important role in molecular recognition because of their capability for specifically binding of a target molecule. A new approach for the separation step has been performed, termed Soluble-SELEX. This new SELEX method uses hybridization as partitioning mechanism for separating the bound and unbound DNA members from the target-molecule. Hybridization procedure has been evaluated by fluorescence studies as partitioning mechanism for SELEX method. Herein, we exploited the incorporation of an aptamer for biosensing detection of a specific target molecule. Three different transduction methods such as fluorescence, electrochemistry and surface plasmon resonance (SPR) were evaluated. In all three cases, the biosensing procedure was successful.In conclusion, this research has evaluated the translation of a fluorescent biosensor into an electrochemical biosensor using maltose-binding protein as biorecognition element. On the other hand, a new SELEX method has been developed. However, future improvements are required in order to optimize the method. As result of SELEX a new avidin-aptamer was selected and three different transduction systems were employed to construct fluorescent, surface Plasmon resonance and electrochemical biosensors. / El presente estudio utiliza dos moléculas diferentes como elementos de bioreconocimiento. En el primer caso, un biosensor basado en proteínas fue desarrollado utilizando la proteína periplasmica de unión a maltosa (MBP = maltose-binding protein). La habilidad para manipular racionalmente la función de una proteína también ofrece la posibilidad de crear nuevas proteínas con valor biotecnológico. Nuestro diseño proteico ha sido usado para evaluar cambios alostéricos en proteínas. Este estudio evalúa un simple cambio conformacional el cual puede ser usado como el principio transductivo para un biosensor. Diferentes estrategias de transducción usando fluorescencia y electroquímica en eventos de reconocimiento entre la proteínas periplasmicas de unión y el ligando, han sido previamente reportadas. Esta investigación inicia con el estudio de los cambios conformacionales de MBP, continuando con el desarrollo de un biosensor electroquímico para maltosa. La señal de cuatro diferentes mutantes (K46C-MBP-MT, N282C MBP-MT, Q72C-MBP-MT; y K25C-MBP-MT) fue evaluada usando voltimetría de onda cuadrada. La posibilidad de usar este tipo de transducción mecánic (distancia) para la configuración de biosensores y la respectiva especificidad analítica es discutida. La segunda parte de este trabajo incluye el método SELEX (systematic evolution of ligands by exponential enrichment) y aptameros como moléculas de bioreconocimiento. Como resultado de el método SELEX, podemos obtener secuencias de oligonucleótidos (aptameros) con propiedades de reconocimiento similares a los anticuerpos. Estos elementos sintéticos, tienen un importante rol en el reconocimiento molecular por su capacidad de unión específica a la molécula blanco. Un nuevo mecanismo para el paso de separación ha sido realizado, y llamado SELEX-Soluble. Este nuevo método SELEX usa la hibridización como mecanismo de separación para dividir los oligonucleótidos de DNA que no se unen y los que se unen a la molécula blanco. El procedimiento de hibridización y su uso como mecanismo de separación en el método SELEX ha sido evaluado a través de estudios de fluorescencia. Este estudio también explora la incorporación de un aptamero como elemento de reconocimiento en un biosensor. Tres diferentes mecanismos de transducción has sido evaluados: fluorescencia, electroquímica y resonancia de plasmon superficial (SPR). En los tres casos una excelente señal fue reportada. En conclusión, esta investigación ha evaluado la transferencia de una biosensor de fluorescencia a un biosensor electroquímico, utilizando la proteína periplásmica de unión a maltosa como elemento de bioreconocimiento. De otro lado, un nuevo método SELEX ha sido desarrollado. Sin embargo, futuras mejoras son requeridas para optimizar el método. Como resultado del método SELEX realizado un nuevo aptamero para avidita ha sido seleccionado y tres diferentes sistemas de transducción ha sido empleado para construir tres diferentes biosensores (fluorescencia, electroquímica y SPR).

Growth and proliferation are normal functions of cells mediated in part via receptor tyrosine kinases such as the epidermal growth factor (EGF) receptor (EGFR). The EGFR binds the extracellular signaling ligand EGF and transduces the signal into the cell. Phosphatidylinositol 3'-kinase (PI3K) responds to EGFR activation and initiates downstream signaling cascades responsible for cell cycle entry, proliferation and inhibition of apoptosis. Cell cycle arrest is required to stop cell growth and proliferation as well as allow apoptosis, if required. The phosphatase and tensin homologue deleted on chromosome ten (PTEN) directly opposes PI3K signaling since its substrate is the PI3K product phosphatidylinositol 3,4,5-trisphosphate. PI3K is a heterodimer composed of a p85 regulatory subunit and a p110 catalytic subunit. PTEN is an essential tumor suppressor protein. Absence of PTEN has been associated with several types of cancer. <p>Our laboratory has characterized new specialized functions for the p85 protein. One function discovered was the ability of p85 to enhance PTEN lipid phosphatase activity. In this thesis PTEN activity is shown to be enhanced at least 3.5 fold in vitro by an equimolar amount of p85. <p>We performed an analysis of PTEN using seven PTEN mutants. Two types of mutants were created: i) regulatory or possible regulatory phosphorylation sites were substituted to mimic both phosphorylated and non-phosphorylated states and ii) alanine substitution of basic amino acid residues. The phosphorylation sites altered were the casein kinase 2 phosphorylation sites in the regulatory domain and tyrosine 336, a proposed regulatory phosphorylation site. Three mutants involving alanine substitution for basic amino acid residues included one mutant in the PASE domain and two more mutants in the C2 domain. It was observed that GFP-PTEN translocates to the plasma membrane upon EGF stimulation. The mimic of constitutive phosphorylation of the Casein kinase 2 sites resulted in cytoplasmic localization whereas the non-phosphorylated mimic was plasma membrane localized regardless of EGFR activation status. Neutralization of positive charge in the PASE and C2 domains seriously impeded the ability of PTEN to bind to phosphorylated phosphatidylinositol lipids and abolished the ability of the protein to translocate to the plasma membrane in response to receptor activation. Located within a cluster of positively charged lysine residues in the C2 domain is a potential phosphorylation site at tyrosine 336. The phosphorylation mimic showed decreased binding to some membrane lipids compared to the non-phosphorylated mimic. The results we generated are consistent with a current model for PTEN regulation that proposes PTEN is localized to the cytoplasm in quiescent cells and dephosphorylation of the regulatory domain occurs upon EGF stimulation allowing translocation to the plasma membrane. The model proposes that dephosphorylation of the casein kinase 2 sites unmasks regions of positive charge that interact with the anionic plasma membrane. Furthermore, the results suggested that at the plasma membrane p85 interacts with PTEN to increase lipid phosphatase activity and may be involved in targeting PTEN to the activated receptor where PI3,4,5P3 lipids are being produced.

Burn induces the inflammation response, and causes the intestinal barrier failure. The failure of intestinal barrier may cause organ damage. Pervious studies have shown that the increase of iNOS activity is closely related to the organ damage after burn. The expression of iNOS is regulated by the activation of NF-£eB, and that is regulated by MAPKs. The pro-inflammatory cytokines play important roles to promote the inflammation through activating a series of signal transduction cascade, via binding to their receptors on cell membrane. The signal transduction cascades are turn on, MAPKs and NF-£eB are activated and the expression of iNOS is promoted. In this study, the role of pro-inflammatory cytokine interleukin-1 receptor (IL-1R) in burn induced intestinal damage was focused on. In experiments, the animals (C57BL/6 mice) were undergone 30~35 % total body surface area (TBSA) burn. The change of intestinal permeability was examined, and intestinal mucosa was assayed for the activation of iNOS and MAPKs by immunoblotting, and the activation of NF-£eB was detected by EMSA. The results reveal that activation of NF-£eB, intestinal permeability and expression of iNOS were increased after burn in wild type mice (WT). ERK MAPK plays an important role to regulate the activation of NF-£eB and expression of iNOS. Surprisingly, the permeability had no change after burn in IL-1R knock out mice (KO). The activation of ERK, NF-£eB and the expression of iNOS were also measured in KO. The levels of p-ERK, NF-£eB activation and iNOS expression were low in KO. When WT mice were treated with U0126 (5 mg/kg i.p.) right after burn to block the activation of ERK, the activation of ERK and NF-£eB, the expression of iNOS, and the intestinal permeability were all decreased significantly. To sum up, the changes in iNOS expression, NF-£eB activation, and intestinal permeability increase are mostly related to the activation of ERK after burn. IL-1 R plays a promotion role in ERK, NF-£eB activation, and iNOS expression that lead to the increase in intestinal permeability and promote damage in intestine.

No description available.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 146-195).

Thesis (M. Sc.)--University of Alberta, 2009. / Title from PDF file main screen (viewed on Nov. 27, 2009). "A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Computing Science, University of Alberta." Includes bibliographical references.