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The short form of dopamine D2 receptor (D2S) functions as a presynaptic autoreceptor on dopamine neurons and has an inhibitory effect on dopaminergic tone. D2-MAPKs pathway is involved in many physiological events like production of prolactin and tyrosine hydroxylase (TH) expression. However, the effect of D2S receptor signalling on MAPKs is cell type specific, and is not fully understood.A recent study in our lab has identified a Gαi-interacting ras-MAPK inhibitor RASA3. Here, we showed that RASA3 is the key effector in D2-induced inhibition of MAPK by knockdown of endogenous RASA3 in the GH4 cell using RASA3 siRNA. We have also transfected a dominant negative RASA3 to compete with the endogenous RASA3 for the binding site on Ras. Both RASA3-siRNA and dominant negative RASA3 blocked D2S-induced inhibition of MAPK activation, clearly implicating that RASA3 is a key effector in Gαi3-dependent D2S mediated MAPKs inhibition To determine whether RASA3’s inhibitory effect could be reconstituted in fibroblast cells, the effect of RASA3 on D2-mediated ERK1/2 activation in COS7 cells was tested. Our results show that both active Gαi2 (or Gαi3) and active RASA3 are required for optimal inhibition of ERK1/2 activation in fibroblast COS7 cells.

Mitochondrial dysfunction is considered as a key mechanism of aging but little is known about the impact of mitochondrial biogenesis. Mitochondrial DNA (mtDNA) copy number control is an important aspect of mitochondrial biogenesis and is highly regulated in eukaryotic organisms. By studying mtDNA copy number, our aim is to gain a better understanding of the relationship between mitochondrial biogenesis and aging. We developed an optimized protocol for measuring mtDNA copy number in Caenorhabditis elegans using quantitative real-time PCR (qPCR). We investigated how mtDNA regulation is affected by a variety of aging-related pathways. We found the insulin/IGF-1 signaling (IIS) pathway regulates mtDNA content in a DAF-16- and UCP-4-dependent manner. By utilizing RNA interference (RNAi) against polg-1, we showed that mitochondrial stress likely modulates lifespan through the IIS pathway. Our work identifies IIS as a communications pathway between mitochondria and the nucleus in modulating mitochondrial biogenesis and lifespan in Caenorhabditis elegans.

The traditional PI3K pathway relies on agonist mediated stimulation of PI3Kα through RTKs and PI3Kγ through GPCRs, which stimulate downstream enzymes such as Akt. This pathway has been found to be important in cardiomyocytes and cardiofibroblasts for regulating cardiac morphology and function. However, evidence has suggested that this traditional pathway does not fully represent the PI3K signaling cascade. We demonstrated that PI3Kγ regulates calcium through kinase independent interactions. PI3KγKO hearts rapidly develop systolic dysfunction and dilated cardiomyopathy in response to pressure overload due to excess matrix metalloproteinase mediated degradation of N-cadherin adhesion complexes. We also show a connection between the PI3K/PTEN and Casein Kinase 2, an enzyme that deactivates PTEN. Finally, our results demonstrate crosstalk between GPCRs and PI3Kα via transactivation of growth factor receptors. Our results provide insight into the regulation and the complexity of the PI3K/PTEN pathway. / Experimental Medicine

The Notch receptor is part of a highly conserved signaling pathway shared in Drosophila, C. elegans and mammals. Extensive studies of Notch signaling have revealed its participation in the development of diverse organ systems including brain, blood cells, blood vessels, gut, and skin. Many genetic modifiers of the Notch signaling pathway have been identified, including some which act at the membrane and others in the nucleus. One such member is Deltex, an E3 ubiquitin ligase, which was originally identified as a modifier of Notch in a Drosophila genetic screen. In early lymphoid development, Deltex has been demonstrated functionally to antagonize Notch signaling but the precise molecular mechanism for this functional antagonism between Notch and Deltex is not understood. However, in Drosophila, recent data supports the formation of a trimeric complex between Deltex, Kurtz and Notch that promotes Notch ubiquitin-mediated proteosomal degradation. Beta-arrestin1 is one of the closest mammalian homologues of Kurtz and functions as an adaptor protein in a variety of cellular processes such as endocytosis, ubiquitination and nuclear shuttling. We hypothesize that a similar interaction occurs in mammalian cells between Notch, beta-arrestin1 and Deltex to negatively modulate the Notch signaling pathway. Our data reveal a physical interaction between beta-arrestin1 and the Notch receptor. We could not, however, detect an interaction between Deltex and beta-arrestin1 by co-immunoprecipitation. We also demonstrate that Notch and beta-arrestin1 physically associate with both a membrane-bound form of activated Notch, as well as the intracellular form of Notch after membrane cleavage. Using RNA interference, as well as overexpression of beta-arrestin1, we demonstrate that beta-arrestin1 negatively regulates a Notch/CSL dependant reporter assay. We also show that the presence of Deltex enhances the negative modulation of the Notch signaling pathway mediated by beta-arrestin1. Therefore, we reveal a new Notch interacting protein and a novel role for beta-arrestin1 in the Notch signaling pathway.

Zugl.: Köln, Univ., Diss., 2006

Bachelor-Arbeit Univ. St. Gallen, 2008.

Modulation of network state is a ubiquitous feature of nervous systems. A major challenge in understanding the physiological flexibility of neural circuits is linking molecules that regulate behaviour to changes in the properties of individual neurons. Here, we use a defined neural circuit in C. elegans to frame this universal problem. By genetic dissection of the behavioural state that sustains escape of 21% O2, we identify novel neuronal functions for several highly conserved genes, including a caspase-like molecule, a calcium-sensitive transcription factor, and two translation initiation factors. These molecules have been implicated in diverse forms of human disease, but their role in the nervous system is either unexplored or poorly understood. Using in vivo Ca2+ imaging techniques to investigate neuron physiology in immobilized and behaving animals, we demonstrate their effect on the properties of individual neurons. The activity of RMG hub neurons is associated with the switch in behavioural state induced by 21% O2. Recently it has been shown that the input-output relationship of RMG is controlled by cytokine signaling, an increasingly appreciated form of neuromodulation. Here I present biochemical and genetic evidence that characterize a novel signaling component downstream of IL-17 receptors in RMG. Our data suggest that, reminiscent of its role in the immune system, it performs both scaffolding and enzymatic functions in neurons. Additionally, we show that RMG responsiveness is controlled by widely expressed, putative regulators of gene expression. Our analyses of these proteins elucidate their function within the URX-RMG circuit, but also raise hypotheses that can be tested more generally in the nervous system. We propose that a calmodulin-binding protein regulates adaptation to ambient O2 conditions, which may reflect a widespread requirement for controlling homeostatic plasticity. Two translation factors that have been shown to be dispensable for general translation are important for regulation of the response to stress. Our study raises the possibility that their role in promoting the activity of all, or some subset of, neurons might underlie this contextual requirement. Together, our findings provide mechanistic insight into the regulation of a behavioural state associated with a specific environmental context.

abstract: The traditional action-response perspective has largely ignored the role of language in competitive dynamics. In this study, I treat language (i.e., word response) as an alternative way to react to rivals when a firm is attacked, in addition to no reaction and action-based reaction. Word response is a specific and public announcement of a focal firm’s potential move in reaction to a competitor’s word or action attack. To explore the underlying mechanism behind word responses, I aim to answer two major questions. The first question is under what situations are responders motivated to use words as competitive responses? For this question I emphasize characteristics of the action, the market, and the actor, using measures such as action type, market dependence of the responder, multimarket contact of the responder in the market, and the competitive aggressiveness of the actor. The second question is what kinds of responders are more likely to use words as competitive responses? For this question, I focus on responder characteristics, such as firm reputation, CEO tenure, and CEO duality. According to Porter’s competitive signaling theory, I argue that responders can use words in reaction to an attack in order to test the waters, deter rivalry, and demonstrate toughness because word responses require few resources, can be accomplished quickly, are reversible, while at the same time still carrying some commitment. Besides incorporating language into the action-response perspective, my dissertation also further integrates the upper-echelons perspective with competitive dynamics research, providing a more realistic and complete understanding of competitive engagement. I test my theory in the consumer electronics (CE) industry with 20 major global CE manufacturers between 2007 and 2014. / Dissertation/Thesis / Doctoral Dissertation Business Administration 2016

There is hardly a cellular process that is not regulated in some way by phosphoinositides, which makes biochemical and physiological studies of these lipids extremely important. PI 3-kinases are key regulators of phosphoinositide metabolism and have been shown to affect a large variety of cellular responses. The key products of PI 3-kinases that have functional activity in higher eukaryotic cells are PI(3,4,5)P₃ and PI(3,4)P₂. PI(3,4,5)P₃ is universally accepted as one of the most important second messengers in signal transduction. However, our knowledge of the functions of PI(3,4)P₂ as a lipid second messenger is much less precise. In this dissertation, work was undertaken to elucidate the regulation of PI(3,4,5)P₃ and PI(3,4)P₂ production and downstream signaling in B cells. Cells with membrane targeted exogenous SHIP were utilized to manipulate phosphoinositide levels. The relationship of PI(3,4,5)P₃ and PI(3,4)P₂ levels to downstream PKB phosphorylation and activation was studied. PI(3,4,5)P₃ and PI(3,4)P₂ levels were found to closely correlate with PKB phosphorylation levels at Thr308 and Ser473, respectively. In addition, PI(3,4)P₂ levels determine the PKB activity in the cytosol; while PI(3,4,5)P₃ levels determine PKB activity at the plasma membrane. Different doses and different forms of B cell receptor (BCR) agonists were used for stimulation. PI 3-kinase activation was studied carefully following stimulation with low doses of anti-BCR antibody and F(ab')₂ fragments. Low concentrations of F(ab')₂ fragments produced higher levels of PI(3,4,5)P₃ than did a high concentration of F(ab')₂ fragments. Downstream PKB signaling was studied in these models. Similar conclusions were drawn from both SHIP over-expressing BJAB cells and dose-dependent BCR stimulations. We speculated that phosphoinositides’ regulation of the kinetics of PKB phosphorylation at Ser473 and Thr308 might be mediated by additional proteins. Investigation of plasma membrane-associated PKB showed that it formed a protein complex of around 400KD, which we attempted to characterize further with respect to PKB phosphorylation and association with lipids. In conclusion, phosphoinositide production is intricately regulated in vivo to control downstream signaling. The levels of PI(3,4)P₂ and PI(3,4,5)P₃ have precise and profound effects on PKB and other molecules such as TAPP and Bam32. This study has contributed new insight into the PI 3-kinase signaling pathway from the aspect of phosphoinositide lipid function. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate