T helper (Th) cell activation and development is one of the most critical events in regulating the adaptive immune response. Understanding its regulation could be of great therapeutical value as many severe diseases are associated with failure in controlling T cell activation and development. However, the regulation of T cell activation appears to be one of the most complex set of cellular and molecular interactions known in the immune system. There is therefore an urgent need for tools to unravel this complexity, and to make use of the quantitative experimental data. To address this issue, mathematical and computational models, based on rigorous biophysical and kinetic data, were developed to study the specific role of some of the major costimulatory molecules involved in Th cell activation, and others developed to investigate proposed theories about mechanisms involved in Th cell differentiation. The simulations of costimulation reveal new implications for the function of the costimulatory molecules CD28 and CTLA-4, and their ligands B7-1 and B7-2, and show how binding affinity, stoichiometric properties, expression levels, and, in particular, competition effects, all profoundly influence complex formation at the immunological synapse. The results support the concept that B7-2 and B7-1 are the dominant ligands of CD28 and CTLA-4, respectively, and indicate that the inability of B7-2 to recruit CTLA-4 to the synapse cannot be, as has been previously proposed, due to the different binding properties of B7-1 and B7-2. Simulations of Th cell development reveal that both instructive and selective processes are likely to be involved in Th cell differentiation. In addition, further simulations indicate that Th2 cells are more likely to become dominant by inhibiting Th1 cells (negative selection), rather than selecting their own growth (positive selection). This thesis also includes an experimental work in which the immunomodulatory role of the bacterial signalling molecule N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL) was analysed. This study strongly suggests that OdDHL suppresses Th cell activation and development, and that it is likely targeting the intracellular signalling events involved in the early stages of Th cell activation.

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Brian G. Rowan. Includes abstract. Document formatted into pages: iv, 204 p. Title from title page of PDF document. Title at ETD Web site: Molecular mechanisms of protein kinase a signaling pathway on estrogen receptor action in breast cancer . Bibliography: pages 59-65, 100-104, 137-150, 167-202.

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Using network architecture to describe a biological system is an effective organizational method. The utility of this approach, which generally applies to qualitative models, is enhanced by the addition of quantitative models characterizing the interactions between network nodes. A chromatophore-based signal transduction network is developed, and the highly interconnected major nodes of the network, guanine trisphosphate, adenylate cyclase, and protein kinase A, are identified. These reference nodes serve to partition the network into functional modules, and mechanistic models describing these modules are derived. Three elicitor compounds, forskolin, melanocyte stimulating hormone (MSH), and clonidine, were selected due to their ability to access the signal transduction network at specific reference nodes, and the module configurations corresponding to their mechanisms of action are presented. The chromatophore responses to the three elicitors and to a negative control, L-15 cell medium, were recorded for two experimental blocks consisting of genetically different fish cells. Significant differences in cell responsiveness were evident between the two blocks, but this variability was controlled by the transformation and normalization of the data. The model parameters for each agent were estimated, and the resulting response curves were highly accurate predictors of the changes in apparent cell area, with R-squared values in the 0.88 to 0.96 range. Two examples were presented for the application of a model discovery algorithm, which selects modules from an existing library, generates model output for all valid module configurations, and selects the configurations which best satisfy a fitness function for a given set of target data. The algorithm proved robust to the introduction of different levels of random error in the simulated data sets when applied to a model of the desensitization of a cell membrane receptor, and continued to classify the stochastic data sets correctly even when the underlying rate constants differed significantly from those embedded in the modules. When challenged with the chromatophore data, the model discovery algorithm successfully matched the forskolin and MSH module configurations to the data within the top three models proposed, with less precise classification for the clonidine model. / Graduation date: 2005

cAMP is a second messenger in a variety of chemosensory receptors, including taste buds and glucose-sensitive pancreatic beta-cells. cAMP is modulated during taste transduction, yet the significance of cAMP changes and the taste cell types in which they occur (Type I glial-like; Type II Receptor; Type III Presynaptic) remain unclear. I developed techniques to image real-time changes in intracellular cAMP in taste cells using genetically-encoded cAMP reporters. This FRET-based reporter permits one to measure single-cell cAMP levels with excellent spatial and temporal resolution (Zaccolo & Pozzan 2002, Science 295:1711). Using a biolistic approach I have transfected rat fungiform taste buds with cAMP reporter plasmids. Focal application of bitter tastant to living fungiform tastebuds in situ produced a decrease in [cAMP]i within individual taste receptor cells. These results are qualitatively similar to previous biochemical measurements from homogenized taste tissue (Yan et al. 2001, Am J Physiol Cell Physiol 280:C742) but are now allowing us to examine the cAMP response in individual, identified cells. I next explored the effect of elevating cAMP on calcium levels, using Fura-2 imaging of isolated mouse vallate taste buds. Elevating [cAMP]i in taste buds evoked calcium responses in presynaptic/Type III taste cells, which do not express GAD1. cAMP induced responses were generated by calcium influx. Using pharmacological antagonists, I determined that the calcium influx triggered by cAMP is through L-type calcium channels, whereas influx following depolarization is primarily through P/Q-type calcium channels. Consistent with these data, single cell RT-PCR showed that the L-type subunit (alpha 1C) was expressed primarily in GAD-negative Presynaptic cells, while the P/Q-type (alpha 1A) was expressed in all Presynaptic cells. Thus, cAMP may modulate the function of synapses in some taste cells. Finally, we have developed a mouse strain expressing a cAMP reporter in a tissue-specific and tetracycline-inducible manner. We crossed this mouse with another strain expressing tet-activator in beta cells of the pancreas. Such islets responded to increasing concentrations of glucose (5.5 to 35mM) with an increase in cAMP levels. The half maximum of 9mM glucose for the cAMP response corresponds well with reported glucose concentrations that elicit insulin release from whole islets. Stimulating pancreatic islets with glucose is known to drive calcium influx into beta-cells. When we simultaneously imaged both second messengers, we found that cAMP changes precede and are independent of calcium changes. In conclusion, these studies have outlined novel potential functions for cAMP signaling in the transduction of both primary tastant and plasma glucose information. In addition, the flexibility of the tet-system will enable cAMP reporter expression in numerous cell types, including those which mediate gustatory transduction.

Chemokines are structurally and functionally related 8-10 kDa proteins defined by four conserved cysteine residues. They consist of a superfamily of proinflammatory mediators that promote the recruitment of various kinds of leukocytes and other cell types through binding to their respective chemokine receptor, a member of the GPCR family. Abnormal control of this system results in various diseases including tumorigenesis and cancer metastasis. Deregulation can occur when constitutively active mutant (CAM) chemokine receptors are locked in the “on” position. This can lead to cellular transformation/tumorigenesis. A viral CAM receptor, ORF74, that can cause tumors in humans, also has homology to human CXC chemokine receptor 2 (CXCR2), which is a G-protein-coupled receptor (GPCR) expressed on neutrophils, some monocytes, endothelial cells, and some epithelial cells. CXCR2 activation with ELR+ CXC chemokines induces leukocyte migration, trafficking, cellular differentiation, angiogenesis and cellular transformation. Using a high throughput yeast screen we identified a novel point mutation, D9H, in CXCR2, which leads to constitutive activation (CA). Generation of positively charged substitutions, D9K and D9R, and D143V as a positive control resulted in CA CXCR2 with differential levels of cellular transformation. To further investigate how D9 mutations lead to differential CA, we used inhibitors of known signal transduction pathways. Pertusiss toxin (PTX) sensitivity in foci formation assays demonstrated that D9R uses the Gi subunit like WTCXCR2 and D143V, while D9H and D9K do not. All CA receptors use the JAK pathway based on sensitivity to the inhibitor, AG490. Phosphorylation of PLC-beta 3 and sensitivity to the PLC-beta 3 inhibitor, U73122, implicates that mutant receptors such as D143V, D9H, D9K, and D9R utilize the Gq/11 subunit. Interestingly, D9R use both Gi and Gq/11 subunits. All of the CA receptors induced phosphorylation of the epidermal growth factor receptor (EGFR) indicating a transactivation between CXCR2 and EGFR. These data describe two novel and important findings. First, N-terminal CXCR2 controls activation and signaling using multiple G protein subunits to elicit downstream signaling. Second, our work supports the “functional selectivity” model for GPCR activation. That is, mimicking agonist activation, CA CXCR2 receptors have multiple conformational states that lead to differential activation.

Environmental signals are sensed by membrane-spanning receptors that communicate with the cell interior. Bacterial chemoreceptors modulate the activity of the CheA kinase in response to binding of small ligands or upon interaction with substrate-bound periplasmic-binding proteins. The mechanism of signal transduction across the membrane is a displacement of the second transmembrane domain (TM2) a few angstroms toward the cytoplasm. This movement repositions a dynamic transmembrane helix relative to the plane of the cell membrane. The research presented in this dissertation investigated the contribution of TM2-membrane interactions to signaling by the aspartate chemoreceptor (Tar) of Escherichia coli. Aromatic residues that reside at the cytoplasmic polar-hydrophobic membrane interface (Trp-209 and Tyr-210) were found to play a significant role in regulating signaling by Tar. These interactions were subsequently manipulated to modulate the signaling properties of Tar. The baseline signaling state was shown to be incrementally altered by repositioning the Trp-209/Tyr-210 pair. To our knowledge, this is the first example of harnessing membrane-protein interactions to modulate the signal output of a transmembrane receptor in a controlled and predictable manner. Potential long-term applications include the use of analogous mutations to elucidate two-component signaling pathways, to engineer the signaling parameters of biosensors that incorporate chemoreceptors, and to predict the movement of dynamic transmembrane helices in silico.

Die adipositasbedingte Insulinresistenz geht mit einer unterschwelligen Entzündungsreaktion einher. Als Antwort auf dieses Entzündungsgeschehen wird PGE2 unter anderem von Kupffer Zellen der Leber freigesetzt und kann seine Wirkung über vier PGE2-Rezeptorsubtypen (EP1-EP4) vermitteln. In vorangegangenen Arbeiten konnte gezeigt werden, dass PGE2 in Rattenhepatozyten über den EP3 R ERK1/2-abhängig die intrazelluläre Weiterleitung des Insulinsignals hemmt. Über die Modulation der Insulinrezeptorsignalkette durch andere EP-Rezeptoren war bisher nichts bekannt. Daher sollte in stabil transfizierten Zelllinien, die jeweils nur einen der vier EP-Rezeptorsubtypen exprimierten, der Einfluss von PGE2 auf die Insulinrezeptorsignalkette untersucht werden. Es wurden HepG2-Zellen, die keinen funktionalen EP-Rezeptor aufwiesen, sowie HepG2-Zellen, die stabil den EP1-R (HepG2-EP1), den EP3β-R (HepG2 EP3β) oder den EP4-R (HepG2 EP4) exprimierten, sowie die humane fötale Hepatozytenzelllinie, Fh hTert, die den EP2- und den EP4-R exprimierte, für die Untersuchungen verwendet. Die Zellen wurden für 330 min mit PGE2 (10 µM) vorinkubiert, um die pathophysiologische Situation nachzustellen und anschließend mit Insulin (10 nM) für 15 min stimuliert. Die insulinabhängige Akt- und ERK1/2-Phosphorylierung wurde im Western-Blot bestimmt. In allen Hepatomzelllinien die EP-R exprimierten, nicht aber in der Zelllinie, die keinen EP R exprimierte, hemmte PGE2 die insulinstimulierte Akt-Phosphorylierung. In allen drei stabil transfizierten Zelllinien, nicht jedoch in den Fh-hTert-Zellen, steigerte PGE2 die basale und insulinstimulierte Phosphorylierung der Serin/Threoninkinase ERK1/2. In den HepG2 EP1- und den HepG2-EP3β-Zellen steigerte PGE2 mutmaßlich über die ERK1/2-Aktivierung die Serinphosphorylierung des IRS, welche die Weiterleitung des Insulinsignals blockiert. Die Hemmung der Aktivierung von ERK1/2 hob in EP3 R-exprimierenden Zellen die Abschwächung der Insulinsignalübertragung teilweise auf. In diesen Zellen scheint die ERK1/2-Aktivierung die größte Bedeutung für die Hemmung der insulinstimulierten Akt-Phosphorylierung zu haben. Da durch die Hemmstoffe die PGE2-abhängige Modulation nicht vollständig aufgehoben wurde, scheinen darüber hinaus aber noch andere Mechanismen zur Modulation beizutragen. In den Fh hTert-Zellen wurde die Insulinrezeptorsignalkette offensichtlich über einen ERK1/2-unabhängigen, bisher nicht identifizierten Weg unterbrochen. Eine gesteigerte PGE2-Bildung im Rahmen der Adipositas ist nicht auf die peripheren Gewebe beschränkt. Auch im Hypothalamus können bei Adipositas Zeichen einer Entzündung nachgewiesen werden, die mit einer gesteigerten PGE2-Bildung einhergehen. Daher wurde das EP R-Profil von primären hypothalamischen Neuronen und neuronalen Modellzelllinien charakterisiert, um zu prüfen, ob PGE2 in hypothalamischen Neuronen die Insulinsignalkette in ähnlicher Weise unterbricht wie in Hepatozyten. In allen neuronalen Zellen hemmte die Vorinkubation mit PGE2 die insulinstimulierte Akt-Phosphorylierung nicht. In der neuronalen hypothalamischen Zelllinie N 41 wirkte PGE2 eher synergistisch mit Insulin. In durch Retinsäure ausdifferenzierten SH SY5Y-Zellen waren die Ergebnisse allerdings widersprüchlich. Dies könnte darauf zurückzuführen sein, dass die Expression der EP Rezeptoren im Verlauf der Kultur stark schwankte und somit die EP R-Ausstattung der Zellen zwischen den Zellversuchen variierte. Auch in den primären hypothalamischen Neuronen variierte die EP R-Expression abhängig vom Differenzierungszustand und PGE2 beeinflusste die insulinstimulierte Akt-Phosphorylierung nicht. Obwohl in allen neuronalen Zellen die Akt-Phosphorylierung durch Insulin gesteigert wurde, konnte in keiner der Zellen eine insulinabhängige Regulation der Expression von Insulinzielgenen (POMC und AgRP) nachgewiesen werden. Das liegt wahrscheinlich an dem niedrigen Differenzierungsgrad der untersuchten Zellen. Im Rahmen der Adipositas kommt es zu einer Überaktivierung des Endocannabinoidsystems. Endocannabinoidrezeptoren sind mit den EP Rezeptoren verwandt. Daher wurde geprüft, ob Endocannabinoide die Insulinsignalweiterleitung in ähnlicher Weise beeinflussen können wie PGE2. Die Vorinkubation der N 41-Zellen für 330 min mit einem Endocannabinoidrezeptoragonisten steigerte die insulinstimulierte Akt-Phosphorylierung, was auf einen insulinsensitiven Effekt von Endocannabinoiden hindeutet. Dies steht im Widerspruch zu der in der Literatur beschriebenen endocannabinoidabhängigen Insulinresistenz, die aber auf indirekte, durch Endocannabinoide ausgelöste Veränderungen zurückzuführen sein könnte. / The obesity related insulin resistance is accompanied by a low grade inflammation. In response to inflammatory stimuli, PGE2 is released from Kupffer cells and signals through four G-Protein coupled PGE2-receptors (EP1-EP4). Previous work showed that PGE2 attenuated insulin signaling in rat hepatocytes through an EP3ß- and ERK1/2-dependent mechanism. Since EP-receptor expression on hepatocytes varies between species and physiological conditions, the effect of the individual EP receptor subtypes on insulin signaling was studied in hepatoma cell lines expressing individual EP receptor subtypes. HepG2 cells lacking functional EP-receptors, and derivatives stably expressing either EP1 receptor (HepG2-EP1), EP3ß receptor (HepG2-EP3ß) or EP4 receptor (HepG2-EP4) and Fh-hTert cells expressing EP2- and EP4-receptor were pre-incubated with PGE2 for 330 min to mimic the sub-acute inflammation. The cells were subsequently stimulated with insulin for 15 min. Akt and ERK1/2 activation was determined by Western Blotting with phospho-specific antibodies. PGE2 inhibited insulin stimulated Akt phosphorylation in all cell lines expressing EP receptors, except in HepG2 cells which are lacking functional EP receptors. PGE2 increased insulin stimulated phosphorylation of the serine/threonine kinase ERK1/2 in all EP R expressing HepG2 cell lines except in Fh-hTert cells. In HepG2-EP1 and HepG2 EP3ß cells PGE2 increased the serine phosphorylation of the insulin receptor substrate, presumably through an ERK1/2 activation. This IRS-serine phosphorylation leads to attenuation of insulin signal transduction. Inhibiting ERK1/2 activation with a specific inhibitor attenuated the PGE2-dependent inhibition of insulin signal transmission in HepG2 EP3ß cells to some extent. ERK1/2 activation in these cells seems to be of major importance for the observed attenuation of insulin stimulated Akt phosphorylation. Application of inhibitors in the other cell lines stably expressing EP receptors provided evidence that other mechanisms contributed to the attenuation of insulin signaling. Insulin signal transduction in Fh-hTert cells by PGE2 was apparently blocked by an ERK1/2-independent mechanism. Increased PGE2 production during obesity is not limited to the periphery. Signs of inflammation have been detected in the hypothalamus, which might be associated with an increased PGE2 production. Therefore, the EP receptor profile of primary neurons as well as neuronal cell models was characterised in order to investigate, whether PGE2 attenuates insulin signal transduction in neuronal cells similar to what was observed in hepatocytes. Pre-incubation with PGE2 did not attenuate insulin stimulated Akt phosphorylation in all neuronal cells. The EP receptor profile in SH SY5Y cells and in primary neurons varied depending on the differentiation status of the cells. Although Akt-kinase was phosphorylated in response to insulin stimulation in all neuronal cells studied, gene expression of insulin target genes (POMC, AgRP) was not modulated by insulin. This might be due to the low level of differentiation of the investigated cells. In the course of obesity, an over-activation of the endocannabinoid system is detected. Since endocannabinoid receptors are related to EP receptors, it was investigated whether endocannabinoids can interfere with insulin signaling in a similar way as PGE2. Pre-incubation of the neuronal cell line N 41 for 330 min with an endocannabinoid receptor agonist, increased insulin stimulated Akt phosphorylation. This implies an insulin sensitising effect of endocannabinoids. This is contradictory to the endocannabinoid-dependent insulin resistance described in the literature and might be caused by indirect endocannabinoid-triggered mechanisms.

Background: Phosphorylation of proteins plays a crucial role in the regulation and activation of metabolic and signaling pathways and constitutes an important target for pharmaceutical intervention. Central to the phosphorylation process is the recognition of specific target sites by protein kinases followed by the covalent attachment of phosphate groups to the amino acids serine, threonine, or tyrosine. The experimental identification as well as computational prediction of phosphorylation sites (P-sites) has proved to be a challenging problem. Computational methods have focused primarily on extracting predictive features from the local, one-dimensional sequence information surrounding phosphorylation sites. Results: We characterized the spatial context of phosphorylation sites and assessed its usability for improved phosphorylation site predictions. We identified 750 non-redundant, experimentally verified sites with three-dimensional (3D) structural information available in the protein data bank (PDB) and grouped them according to their respective kinase family. We studied the spatial distribution of amino acids around phosphorserines, phosphothreonines, and phosphotyrosines to extract signature 3D-profiles. Characteristic spatial distributions of amino acid residue types around phosphorylation sites were indeed discernable, especially when kinase-family-specific target sites were analyzed. To test the added value of using spatial information for the computational prediction of phosphorylation sites, Support Vector Machines were applied using both sequence as well as structural information. When compared to sequence-only based prediction methods, a small but consistent performance improvement was obtained when the prediction was informed by 3D-context information. Conclusion: While local one-dimensional amino acid sequence information was observed to harbor most of the discriminatory power, spatial context information was identified as relevant for the recognition of kinases and their cognate target sites and can be used for an improved prediction of phosphorylation sites. A web-based service (Phos3D) implementing the developed structurebased P-site prediction method has been made available at http://phos3d.mpimp-golm.mpg.de.

Plasmin, the central molecule of the plasminogen activator system, is a broad-spectrum serine protease. Plasmin is important for the degradation of fibrin and other components of the extracellular matrix (ECM) during a number of physiological and pathological processes. The aim of this thesis was to elucidate the functional roles of plasmin during pathological inflammation and infection in autoimmune and non-autoimmune diseases. For this purpose, mouse models of rheumatoid arthritis (RA), bacterial arthritis, infection, and sepsis have been used. Previous studies from our laboratory have shown that plasminogen-deficient mice are resistant to the development of collagen type II-induced arthritis (CIA). In contrast, others have shown that plasmin plays a protective role in antigen-induced arthritis (AIA). To investigate the contrasting roles of plasminogen deficiency in models of CIA and AIA, a new animal model of arthritis called local injection-induced arthritis (LIA) was developed. In this model, we replaced methylated bovine serum albumin, which is normally used as an immunogen in the AIA model, with collagen type II (CII) to induce arthritis. When wild-type and plasminogen-deficient mice were injected intra-articularly with CII or 0.9% NaCl following CIA induction, plasminogen-deficient mice developed typical CIA, but the disease was less severe than in wild-type mice and was restricted to the injected joints. When the AIA model was used, plasminogen-deficient mice developed a much more severe arthritis than the wild-type mice. These results indicate that both the antigen and joint trauma caused by the local injection are critical to explaining the contrasting roles of plasminogen deficiency in CIA and AIA. This indicates that CIA and AIA have distinct pathogenic mechanisms and plasmin plays contrasting roles in different types of arthritis models. To study the functional roles of plasmin in the host inflammatory response during infectious arthritis, a Staphylococcus aureus-induced bacterial arthritis model was established. When wild-type mice were injected intra-articularly with 1 × 106 colony-forming units (CFU) of S. aureus per joint, all the bacteria were completely eliminated from the injected joints in 28 days. However, in the plasminogen-deficient mice, the S. aureus counts were 27-fold higher at day 28 than at day 0. When human plasminogen was given to the plasminogen-deficient mice daily for 7 days, the bacterial clearance was greatly improved and the necrotic tissue in the joint cavity was also completely eliminated. Supplementation of plasminogen-deficient mice with plasminogen also restored the expression level of interleukin-6 (IL-6) in the arthritic joints. In summary, plasmin has protective roles during S. aureus-induced arthritis by enhancing cytokine expression, removing necrotic tissue, and mediating bacterial killing and inflammatory cell activation. The functional roles of plasmin during infection and sepsis were also studied in mice. Infection was induced by injecting 1 × 107 CFU of S. aureus intravenously and the sepsis model was induced by injecting 1.6 × 108 CFU of S. aureus. In the infection model, the wild-type mice had a 25-day survival rate of 86.7%, as compared to 50% in the plasminogen-deficient group. However, when sepsis was induced, the average survival for plasminogen-deficient mice was 3 days longer than for wild-type mice. Twenty-four hours after the induction of sepsis, the serum levels of IL-6 and IL-10 as well as the bacterial counts in all organs investigated were significantly higher in wild-type mice than in plasminogen-deficient mice. In wild-type mice, blockade of IL-6 by intravenous injection of anti-IL-6 antibodies significantly prolonged the onset of mortality and improved the survival rate during sepsis. These data indicate that plasmin plays different roles during infection and sepsis. Furthermore, plasmin appears to be involved in the regulation of inflammatory cytokine expression during sepsis. Taken together, our data indicate that plasmin plays multifunctional pro-inflammatory roles in different autoimmune and non-autoimmune diseases. The pro-inflammatory roles of plasmin include activation of inflammatory cells, regulation of cytokine expression, and enhancement of the bacterial killing ability of the host.