Molekulare Grundlagen der PI3-Kinase-γ-Regulation

Title and Contents I 1 Introduction 1 2 Aims 23 3 Materials and methods 25 4 Results 53 5 Discussion 103 6 Summary 125 7 Zusammenfassung 127 References 129 Appendix 159Phosphoinositide 3-kinase γ (PI3Kγ) is a lipid kinase that is chiefly activated downstream of G protein-coupled receptors. By producing the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) it addresses a plethora of effector proteins that contain PIP3-binding pleckstrin homology domains. By virtue of these effectors, PI3Kγ controls many cellular processes ranging from cell survival and proliferation to chemotaxis and production of reactive oxygen species. PI3Kγ consists of a catalytic p110γ and a regulatory p101 subunit. p101 binds to Gβγ complexes released from heterotrimeric G proteins and thereby co-translocates the bound catalytic p110γ subunit to the plasma membrane, where it has access to its lipid substrate. Gβγ and Ras proteins may further stimulate catalytic activity by allosteric mechanisms. Despite their crucial role in the activation of p110γ, the binding sites on p101 for p110γ and Gβγ remained largely elusive. Therefore, this thesis primarily aimed at elucidating the elements within the primary structure of p101 that are responsible for interaction with p110γ and Gβγ. To this end, deletion mutants of p101 were constructed and analyzed with respect to their ability to bind p110γ and Gβγ. The heterodimerization domain could be ascribed to an N-terminal region (residues 25-175), whereas the Gβγ binding domain resides within the C terminus of p101 (residues 650-850). These domains were found to be highly conserved between orthologues of p101, whereas the intervening sequence stretches display a less stringent conservation. Strikingly, data base searches performed to identify novel p110γ\- or Gβγ- binding proteins revealed a cDNA that shows significant similarity to p101 within the p110γ\- and Gβγ-binding regions. Cloning of the corresponding coding sequence from dendritic cells and subsequent characterization of the encoded protein yielded a novel regulatory subunit of PI3Kγ. According to its molecular weight and function, it was termed PI3Kγ adapter protein of 87 kDa (p87PIKAP). p87PIKAP interacts with both p110γ and Gβγ, thereby, like p101, mediating activation of p110γ downstream of GPCR stimulation. At least one of these regulatory subunits accompanies p110γ in all cell types assayed. Whereas expression of p101 dominates in B and T cells, both subunits can be found in dendritic cells, macrophages, and neutrophils. In heart and mast cells, however, p87PIKAP is expressed almost exclusively. In the mast cell-related cell line RBL-2H3, knockdown of p87PIKAP expression by shRNA led to diminished PI3Kγ activity and degranulation. Thus, the p110γ- dependent feedback loop, which amplifies antigen-induced degranulation via autocrine stimulation of Gi-coupled GPCRs, is dependent on p87PIKAP. A negative inotropic impact of PI3Kγ on cardiac contractility has recently been shown to be mediated by stimulation of phosphodiesterase 3B within a macromolecular complex that contains p110γ as an essential component. However, p110γ is itself unable to bind PDE3B. p87PIKAP and p110γ/p87PIKAP heterodimers were found to copurify with PDE3B from co-transfected cells, indicating that p87PIKAP may be an essential component of the PDE3B regulatory complex.Die Phosphatidylinositol-3-Kinase γ (PI3Kγ) ist eine Lipidkinase, die vor allem nach Stimulation von G-Protein-gekoppelten Rezeptoren aktiviert wird. Der von ihr produzierte sekundäre Botenstoff Phosphatidylinositol-3,4,5-trisphosphat (PIP3) aktiviert zahlreiche Effektorproteine, die PIP3-bindende Pleckstrin-Homologie-Domänen aufweisen. Über diese Effektoren kontrolliert PI3Kγ zahlreiche zelluläre Prozesse wie das Zellüberleben, die Proliferation oder zytoskelettale Veränderungen. PI3Kγ besteht aus der katalytischen p110γ-Untereinheit und der regulatorischen p101-Untereinheit, die für die Aktivierung der PI3Kγ essentiell ist. p101 bindet Gβγ-Komplexe und transloziert so die p110γ-Untereinheit zur Plasmamembran, wo sich ihr Lipidsubstrat befindet. Dort wird p110γ ebenfalls durch Gβγ, aber auch durch Ras, zusätzlich allosterisch stimuliert. Trotz der Relevanz der von p101 vermittelten Interaktionen blieben ihre Bindestellen für p110γ und Gβγ noch weitgehend ungeklärt. Daher war es das Ziel dieser Arbeit, zunächst die Elemente in der Primärstruktur der p101 zu ermitteln, die für diese Interaktionen verantwortlich sind. Zu diesem Zweck wurden p101-Deletionsmutanten erstellt und auf ihre Bindung zu p110γ und Gβγ hin untersucht. Die p110γ-bindende Heterodimerisierungsdomäne konnte dabei auf die Aminosäuren 25 175 eingegrenzt werden, während die Gβγ- Bindedomäne im C-Terminus der p101 liegt (Aminosäuren 650 850). Diese Domänen sind bei Orthologen der p101 hoch konserviert, während die übrigen Bereiche weniger streng konserviert sind. In Datenbank-Suchen nach potenziellen neuen p110γ-Interaktionspartnern und unbekannten Gβγ-Effektoren wurde erstaunlicherweise eine cDNA-Sequenz identifiziert, die innerhalb der Interaktionsdomänen deutliche Ähnlichkeit zu p101 aufwies. Die Klonierung der kodierenden Sequenz aus dendritischen Zellen und die nachfolgende Charakterisierung des kodierten Proteins bestätigten, dass es sich um eine neue regulatorische PI3Kγ-Untereinheit handelt. Sie wurde als 87 kDa PI3Kγ-Adapterprotein bezeichnet (p87PIKAP). p87PIKAP interagiert mit p110γ und Gβγ und ist so in der Lage, wie p101 die Aktivierung der p110γ zu vermitteln. Mindestens einer dieser beiden Adaptoren ist in allen untersuchten p110γ-exprimierenden Zelltypen zu finden. Während in B- und T-Zellen p101 überwiegt, finden sich beide Untereinheiten in dendritischen Zellen, Makrophagen und Neutrophilen. Im Herzen und in Mastzellen hingegen ist fast ausschließlich p87PIKAP vorhanden. In der Mastzell-verwandten Zelllinie RBL-2H3 führte eine shRNA-vermittelte Reduktion der p87PIKAP-Expression zu einer stark verminderten PI3Kγ-Aktivität und einer deutlich reduzierten Degranulation. Die p110γ-vermittelte Verstärkung der Degranulation, die über autokrine Stimulation von Gi- gekoppelten Rezeptoren verläuft, ist also von p87PIKAP abhängig. Eine negativ inotrope Wirkung der PI3Kγ im Herzen konnte kürzlich einer Stimulation der Phosphodiesterase 3B durch p110γ zugewiesen werden, die auf der Bildung eines Proteinkomplexes beruht. Jedoch kann p110γ alleine diese Interaktion nicht ausbilden. Hingegen konnte p87PIKAP alleine und in Komplex mit p110γ mit PDE3B kopräzipitiert werden. Daher könnte p87PIKAP ebenfalls ein essentieller Bestandteil des PDE3B-regulierenden Komplexes sein

Simulation and measurement of the water-sided viscous shear stress without waves

The local viscous sheer stress in the watersided boundary layer of the air-water interface was measured at the Aeolotron, an annular wind-wave facility located at the Institute of Environmental Physics in Heidelberg. The measurements were conducted at low wind speeds up to u_10 ~ 6.1 m/s, under non-stationary conditions directly after turning the wind on, while periodically switching the wind on and off, as well as under stationary conditons with the wind- and water flow fields being in equilibrium. The wave formation was suppressed by adding 3.3 mg/L of the surfactant Triton X-100 to the water. Active thermography was used for heating thin lines on the water surface, and monitoring their broadening. This method combines the advantages of an air-sided and contactless setup and independence on the prevailing wind profile. Using an approach based on simulation developed in this thesis, the viscous shear stress could be obtained with a maximal temporal resolution of one second. Thereby the temporal development of the line widths were simulated numerically, varying three parameters. These were the initial line width and the spatially constant components of the shear stress with respect to depth and the direction parallel to the lines. The results of the simulations were interpolated and matched with the measured line widths. The hereby obtained values for the viscous shear stress were for the stationary measurements compared to a direct measurement based on particle streak velocimetry. The discrepancies were smaller than 20 %

Timing Analysis of Embedded Software Updates

We present RETA (Relative Timing Analysis), a differential timing analysis technique to verify the impact of an update on the execution time of embedded software. Timing analysis is computationally expensive and labor intensive. Software updates render repeating the analysis from scratch a waste of resources and time, because their impact is inherently confined. To determine this boundary, in RETA we apply a slicing procedure that identifies all relevant code segments and a statement categorization that determines how to analyze each such line of code. We adapt a subset of RETA for integration into aiT, an industrial timing analysis tool, and also develop a complete implementation in a tool called DELTA. Based on staple benchmarks and realistic code updates from official repositories, we test the accuracy by analyzing the worst-case execution time (WCET) before and after an update, comparing the measures with the use of the unmodified aiT as well as real executions on embedded hardware. DELTA returns WCET information that ranges from exactly the WCET of real hardware to 148% of the new version's measured WCET. With the same benchmarks, the unmodified aiT estimates are 112% and 149% of the actual executions; therefore, even when DELTA is pessimistic, an industry-strength tool such as aiT cannot do better. Crucially, we also show that RETA decreases aiT's analysis time by 45% and its memory consumption by 8.9%, whereas removing RETA from DELTA, effectively rendering it a regular timing analysis tool, increases its analysis time by 27%

Spiral Growth and Step Edge Barriers

The growth of spiral mounds containing a screw dislocation is compared to the growth of wedding cakes by two-dimensional nucleation. Using phase field simulations and homoepitaxial growth experiments on the Pt(111) surface we show that both structures attain the same characteristic large scale shape when a significant step edge barrier suppresses interlayer transport. The higher vertical growth rate observed for the spiral mounds on Pt(111) reflects the different incorporation mechanisms for atoms in the top region and can be formally represented by an enhanced apparent step edge barrier.Comment: 11 pages, 4 figures, partly in colo

Taxonomy and analysis of issues facing post mission disposal concept

In order to ensure a sustainable space environment for future generations a strategy for all spacefarers must be developed in order to mitigate the growth of the space debris population. To this end, this preliminary analysis is the first step towards the development of a cost-efficient but highly reliable PMD (Post Mission Disposal) module. This PMD module will be attached to the spacecraft on ground and will ensure the removal of the spacecraft at the end of the nominal operational lifetime or act as a removal back-up in the case of loss of control of the spacecraft. The PMD module will be scalable and flexible, enabling the PMD of any future spacecraft in an Earth orbit. Ultimately, the gap between the 90% PMD success rate required by ISO 24113:2011(E) and the current success rate of 50%-60% can be closed. A survey of de- and re-orbit techniques and concepts was carried out and a taxonomy of approximately 40 concepts, including 12 which do not appear in the literature, is presented. A qualitative analysis was carried out on the concepts identified in the taxonomy, and a comparison matrix was built including 12 different comparison metrics. The 5 most promising concepts for the PMD module were down-selected from this matrix. These concepts were: drag augmentation, solar sailing, electrodynamic tether, low thrust propulsion and high thrust propulsion. A further 3 additional concepts were also defined by considering combinations of the down-selected concepts. A quantitative analysis of the down-selected concepts was performed using a purpose built analytical analysis tool. This tool was designed to rapidly predict re-entry epochs of space objects, given specific mission parameters. The analytical nature of this tool allowed for a Monte Carlo analysis, resulting in trade-off analyses within and between the different concepts for various mission parameters. The output of the quantitative analysis provided preliminary mission parameters, systems sizing and trade-off data on each of the down-selected concepts and combination concepts. From this analysis it was concluded that each system had its advantages, and challenges, so recommendations were made on how each system could be used to its maximum potential and which systems were more effective than others in specific situations. The most prominent of these results were the need for the PMD to de-tumble the spacecraft prior to deployment of the removal system, and the fact that none of the down-selected concepts were recommended for use in long term missions

iDISCO+ for the Study of Neuroimmune Architecture of the Rat Auditory Brainstem

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei

Morphological stability of electromigration-driven vacancy islands

The electromigration-induced shape evolution of two-dimensional vacancy islands on a crystal surface is studied using a continuum approach. We consider the regime where mass transport is restricted to terrace diffusion in the interior of the island. In the limit of fast attachment/detachment kinetics a circle translating at constant velocity is a stationary solution of the problem. In contrast to earlier work [O. Pierre-Louis and T.L. Einstein, Phys. Rev. B 62, 13697 (2000)] we show that the circular solution remains linearly stable for arbitrarily large driving forces. The numerical solution of the full nonlinear problem nevertheless reveals a fingering instability at the trailing end of the island, which develops from finite amplitude perturbations and eventually leads to pinch-off. Relaxing the condition of instantaneous attachment/detachment kinetics, we obtain non-circular elongated stationary shapes in an analytic approximation which compares favorably to the full numerical solution.Comment: 12 page

Transport Coefficients from Large Deviation Functions

We describe a method for computing transport coefficients from the direct evaluation of large deviation function. This method is general, relying on only equilibrium fluctuations, and is statistically efficient, employing trajectory based importance sampling. Equilibrium fluctuations of molecular currents are characterized by their large deviation functions, which is a scaled cumulant generating function analogous to the free energy. A diffusion Monte Carlo algorithm is used to evaluate the large deviation functions, from which arbitrary transport coefficients are derivable. We find significant statistical improvement over traditional Green-Kubo based calculations. The systematic and statistical errors of this method are analyzed in the context of specific transport coefficient calculations, including the shear viscosity, interfacial friction coefficient, and thermal conductivity.Comment: 11 pages, 5 figure

The 3′ processing of antisense RNAs physically links to chromatin-based transcriptional control

Noncoding RNA plays essential roles in transcriptional control and chromatin silencing. At Arabidopsis thaliana FLC, antisense transcription quantitatively influences transcriptional output, but the mechanism by which this occurs is still unclear. Proximal polyadenylation of the antisense transcripts by FCA, an RNA-binding protein that physically interacts with RNA 3′ processing factors, reduces FLC transcription. This process genetically requires FLD, a homolog of the H3K4 demethylase LSD1. However, the mechanism linking RNA processing to FLD function had not been established. Here, we show that FLD tightly associates with LUMINIDEPENDENS (LD) and SET DOMAIN GROUP 26 (SDG26) in vivo, and, together, they prevent accumulation of monomethylated H3K4 (H3K4me1) over the FLC gene body. SDG26 interacts with the RNA 3′ processing factor FY (WDR33), thus linking activities for proximal polyadenylation of the antisense transcripts to FLD/LD/SDG26-associated H3K4 demethylation. We propose this demethylation antagonizes an active transcription module, thus reducing H3K36me3 accumulation and increasing H3K27me3. Consistent with this view, we show that Polycomb Repressive Complex 2 (PRC2) silencing is genetically required by FCA to repress FLC. Overall, our work provides insights into RNA-mediated chromatin silencing