Studien an signalleitenden Adapterproteinen in wildtypischen und virusinfizierten Lymphocyten

Engels N. Studien an signalleitenden Adapterproteinen in wildtypischen und virusinfizierten Lymphocyten. Bielefeld (Germany): Bielefeld University; 2005.Das Immunsystem der Wirbeltiere ist ein aus verschiedenartigen Zellen bestehendes System, das den Organismus vor Krankheitserregern schützt. Im Falle einer Infektion vermag es die Erreger meist zu eliminieren und dem Individuum anschließend einen langanhaltenden Schutz vor einer erneuten Infektion mit dem gleichen Erreger zu verleihen. Das Immunsystem wird im wesentlichen von Leukocyten gebildet, von denen B- und T-Lymphocyten das sog. adaptive Immunsystem darstellen. B- und T-Lymphocyten besitzen hochspezifische Rezeptoren, die an Pathogene oder deren Bestandteile (allg. Antigene) binden können. Diese Antigenrezeptoren lösen durch Bindung an ein Antigen Signale im Inneren der Zelle aus, die für deren Aktivierung und die darauffolgende Initiation einer Immunantwort notwendig sind. Das cytoplasmatische Adapterprotein SLP-65 ist für B-Zell-Antigenrezeptor-(BCR-)vermittelte Signalleitungsereignisse essentiell, da es signalleitende Enzyme und deren Substrate in multimolekularen Komplexen organisiert. Ohne diese Organisation verläuft die Aktivierung nachfolgender biochemischer Prozesse ineffizient oder bleibt gänzlich aus, was beim Menschen eine schwere Immundefizienz zur Folge hat. Die im Rahmen dieser Arbeit durchgeführten biochemischen Untersuchungen zur Funktion von SLP-65 führten zu der Entdeckung, dass SLP-65 über seine SH2-Domäne direkt mit der BCR-Komponente Ig-[alpha] interagiert. Diese Interaktion erfolgt über eine außerhalb des ITAMs gelegene, bis dato unbekannte Phosphorylierungsstelle in Ig-[alpha], Tyrosin 204 (Y 204). Des weiteren konnte demonstriert werden, dass SLP-65 essentiell für die Funktion des Latenten Membranproteins 2A (LMP2A), eines Proteins des Epstein-Barr Virus (EBV), ist. Das EBV infiziert selektiv B-Lymphocyten, in denen es einen lebenslangen latenten Infektionszustand etablieren kann. LMP2A ist an der Aufrechterhaltung der Latenz beteiligt und bedient sich zu diesem Zweck, wie in dieser Arbeit gezeigt wird, des SLP-65-Proteins. Ein weiteres Ziel dieser Arbeit war die Identifizierung neuer signalleitender Proteine in Lymphocyten. Mittels Recherche in Sequenzdatenbanken konnten zwei neue Gene identifiziert und deren cDNAs kloniert werden. Bei den davon abgeleiteten Proteinen, p120 und NBP64, handelt es sich vermutlich um Adapterproteine, da bekannte katalytische Domänen nicht vorhanden sind. p120 weist Homologie zu den hämatopoetischen Adapterproteinen ADAP und PRAM-1 auf. Ferner assoziiert es wie diese in vitro mit Proteinen der SKAP-Familie. NBP64 enthält eine BEACH-Domäne und mehrere WD40-repeats, sowie vermutlich eine PH-ähnliche Domäne. Die Funktion aller dieser Domänen ist unklar, jedoch sind keine BEACH-Domänen oder WD40-repeats enthaltenden Proteine mit katalytischer Aktivität bekannt. Während die Expression von p120 nicht abschließend geklärt ist, wird NBP64 präferentiell in lymphatischem Gewebe exprimiert. Zusammengenommen werden in dieser Arbeit neue Eigenschaften des BCRs und des Adapterproteins SLP-65 in normalen und EBV-infizierten B-Zellen beschrieben. Des weiteren werden zwei neue Adapterproteine, p120 und NBP64, vorgestellt

Yield stress distribution in injection-mouldedglassy polymers

A methodology for structural analysis simulations is presented that incorporates the distribution of mechanical propertiesalong the geometrical dimensions of injection-moulded amorphous polymer products. It is based on a previously developedmodelling approach, where the thermomechanical history experienced during processing was used to determine the yield stressat the end of an injection-moulding cycle. Comparison between experimental data and simulation results showed an excellentquantitative agreement, both for short-term tensile tests as well as long-term creep experiments over a range of strain rates,applied stresses, and testing temperatures. Changes in mould temperature and component wall thickness, which directly affectthe cooling profiles and, hence, the mechanical properties, were well captured by the methodology presented. Furthermore, itturns out that the distribution of the yield stress along a tensile bar is one of the triggers for the onset of the (strong) localizationgenerally observed in experiments.Spanish Government (Ministry of Sci-ence and Innovation, Ministry of Economy and Competitiveness)through grant numbers RYC-2010-07171 and DPI2011-25470This is the peer reviewed version of the following article: Verbeeten, W. M., Kanters, M. J., Engels, T. A. and Govaert, L. E. (2015), Yield stress distribution in injection-moulded glassy polymers. Polym. Int., 64(11): 1527–1536, which has been published in final form at http://dx.doi.org/10.1002/pi.4898. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archivin

The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions

Non–T Cell Activation Linker (NTAL): A Transmembrane Adaptor Protein Involved in Immunoreceptor Signaling

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcɛ-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells

Anisotropic rate-dependent mechanical behavior of Poly(Lactic Acid) processed by Material Extrusion Additive Manufacturing

The strain-rate dependence of the yield stress for Material Extrusion Additive Manufacturing (ME-AM) polylactide samples was investigated. Apparent densities of the ME-AM processed tensile test specimens were measured and taken into account in order to study the effects of the ME-AM processing step on the material behavior. Three different printing parameters were changed to investigate their influence on mechanical properties, i.e. infill velocity, infill orientation angle, and bed temperature. Additionally, compression molded test samples were manufactured in order to determine bulk properties, which have been compared to the ME-AM sample sets. Anisotropy was detected in the strain-rate dependence of the yield stresses. ME-AM samples with an infill angle of 0° have a higher strain-rate dependence than specimens with αor = 90°. Remarkably, the strain-rate dependence manifested by the ME-AM samples is considerably lower than that displayed by compression molded test specimens. The Ree-Eyring modification of the Eyring flow rule is able to accurately describe the strain-rate dependence of the yield stresses, taking two molecular deformation processes into account to describe the yield kinetics. The results from this paper further show a change from a brittle behavior in case of compression molded samples to a semi-ductile behavior for some of the ME-AM sample sets. This change is attributed to the processing phase and stresses the importance that the temperature profile (initial fast cooling combined with successive heating cycles) and the strain profile during ME-AM processing have on the resulting mechanical properties. Both these profiles are significantly different from the thermo-mechanical history that material elements experience during conventional processing methods, e.g. injection or compression molding. This paper can be seen as initial work that can help to further develop predictive numerical tools for Material Extrusion Additive Manufacturing, as well as for the design of structural components

The Rhetoric of Multilateral Foreign Aid. Assessing the Importance of Good Governance as a Lending Criterion of the World Bank

This study presents two areas of good governance and assesses their relevance for the World Bank's lending policy. First, the definitions and criteria of good governance of four multilateral organisations, namely the World Bank, the International Monetary Fund, the United Nations Development Program and the Development Assistance Committee of the OECD are outlined and discussed. A differentiation between a non-political core area and an associated political area of governance is introduced, and the different governance aspects are allocated to them. Building on these findings, the second part of this study analyses the importance of good governance and alternative factors as lending criteria for the World Bank. Several OLS regressions for three cross-sections between 1983 and 1997 show that the different aspects of good governance play only a subordinate role for the allocation of World Bank credits. Only countries reducing their excessive military potential are favoured by the World Bank shortly after the end of the Cold War. Instead, economic and other donor interests, such as length of colonial dependence, increasingly influence the Bank's credit lending policy over time. The results show little statistical evidence for an impact of recipient need considerations. As previous literature has shown, a negative relationship between population size of a country and the share and change of lending is also observed. Further, the World Bank and the International Monetary Fund focus on the same developing countries, whereas the official development assistance of the U.S. is directed to different countries