Klonierung und in vivo Analyse von Proteinen des menschlichen inneren Kinetochors

Die korrekte Trennung der Schwesterchromatiden während der Mitose ist unumgänglich für die Stabilität des Genoms und somit ein Grundpfeiler des Lebens. Um diese Herausforderung zu bewältigen, besitzt jedes Chromosom an der primären Einschnürungsstelle einen Proteinkomplex, das Kinetochor, welches die Anheftung der Mikrotubuli und die Regulation der Segregation gewährleistet. Im Rahmen dieser Arbeit konnte gezeigt werden, dass der Kinetochorkomplex allerdings nicht nur während der Mitose essentielle Aufgaben zu erfüllen hat, sondern auch innerhalb der Interphase einer Vielzahl von Umbauprozessen durchläuft und spezifische Funktionen realisiert. Erstmals wurde eine detaillierte Analyse der dynamischen Eigenschaften, Bindungsverhältnisse, Proteingehalte und Nachbarschaften eines der inneren Kinetochorelemente, CENP-N, über den Zellzyklus hinweg durchgeführt. Diese Untersuchungen erfolgten in lebenden menschlichen Zellen mittels hochauflösender Mikroskopie und zeichnen so ein realistisches Bild von den Vorgängen am Kinetochor. Sie ermöglichen Rückschlüsse auf die Funktionen von CENP-N, welches daraufhin als Genauigkeitsfaktor für den Einbau von CENP-A postuliert wurde. Desweiteren deuten die Ergebnisse darauf hin, dass CENP-N eine Markierung des Kinetochors während der Replikation der centromerischen DNA sein könnte. Diese Herangehensweise zum Studium der Eigenschaften wurde auf weitere Proteine des inneren Kinetochors, wie CENP-T und –W sowie den O/P/Q/R/U-Komplex, übertragen. Basierend darauf stellte sich heraus, dass das Kinetochor im Verlauf der Interphase zwei Hauptaufgaben zu erfüllen hat. Hierbei liegt der Schwerpunkt während der ersten Hälfte der Interphase auf der Erhaltung der Kinetochorstruktur selbst. Grundlage ist der korrekte Einbau von CENP-A in den centromerischen Lokus, welcher unter anderem von CENP-C und CENP-I bewerkstelligt wird. Gleichzeitig kommt er zu einem zeitweiligen Umbau der zugrunde liegenden Chromatinstruktur. Nach Abschluss dieses Vorgangs werden während der zweiten Hälfte der Interphase sukzessive alle weiteren Komponenten des CCAN neu rekrutiert und es bildet sich eine Plattform für die Anlagerung des äußeren Kinetochorproteine, welche die eigentliche Trennung der Chromatiden ermöglichen. Dieses Fundament wird verankert durch CENP-C und CENP-T. Erst nach dem Ablauf dieser Prozesse ist das centromerische Chromatin bereit für die Mitose.The accurate division of sister chromatides during mitosis is essential for stability of the genome and therefore a keystone of life. To tackle this challenge each chromosome contains a protein complex at the primary constriction, called kinetochore, which facilitates the attachment of microtubules and the regulation of segregation. This dissertation shows that the kinetochore complex executes essential tasks not only during mitosis but also undergoes several rearrangements und realizes specific functions in the course of the interphase. For the first time, the dynamic behavior, binding properties, protein content and neighborhood relationships of a kinetochore protein, CENP-N, were analyzed in detail throughout the cell cycle. These examinations were performed in living cells via high resolution microscopy, which procure a realistic picture of kinetochore-associated processes. The results provide evidence that CENP-N functions as a fidelity factor for CENP-A loading at the kinetochore. Furthermore, the findings suggest that CENP-N marks the kinetochore region during replication of centromeric chromatin. This approach was then used to investigate characteristics of other proteins of the inner kinetochore, e.g. CENP-T and –W as well as the O/P/Q/R/U complex. Based on the obtained results the kinetochore has to achieve two main functions. During the first half of the cell cycle, the inner kinetochore is maintained in its structure. This depends on the accurate assembly of CENP-A into the centromeric locus facilitated by CENP-C and CENP-I. In parallel, a temporary rearrangement of centromeric chromatin takes place. After completion of this process, during second half of interphase other components of CCAN are successive recruited and build a platform for attachment of outer kinetochore proteins to allow proper division of chromatides. This fundament is anchored by CENP-C and CENP-T. After this event the centromeric chromatin is switched to a mitotic state

Step-wise assembly, maturation and dynamic behavior of the human CENP-P/O/R/Q/U kinetochore sub-complex

Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment

Fc receptor-mediated immunity against Bordetella pertussis

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcγRIIa (CD32) and FcγRIIIb (CD16), working synergistically. Furthermore, these FcγR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcαR (CD89). Simultaneous engagement of FcαRI and FcγR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Fc receptor-mediated immunity against Bordetella pertussis

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcγRIIa (CD32) and FcγRIIIb (CD16), working synergistically. Furthermore, these FcγR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcαR (CD89). Simultaneous engagement of FcαRI and FcγR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.Centro de Investigación y Desarrollo en Fermentaciones Industriale

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt–Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS

Fc receptor-mediated immunity against Bordetella pertussis

The relevance of specific Abs for the induction of cellular effector functions against Bordetella pertussis was studied. IgG-opsonized B. pertussis was efficiently phagocytosed by human polymorphonuclear leukocytes (PMN). This process was mediated by the PMN IgG receptors, FcγRIIa (CD32) and FcγRIIIb (CD16), working synergistically. Furthermore, these FcγR triggered efficient PMN respiratory burst activity and mediated transfer of B. pertussis to lysosomal compartments, ultimately resulting in reduced bacterial viability. Bacteria opsonized with IgA triggered similar PMN activation via FcαR (CD89). Simultaneous engagement of FcαRI and FcγR by B. pertussis resulted in increased phagocytosis rates, compared with responses induced by either isotype alone. These data provide new insights into host immune mechanisms against B. pertussis and document a crucial role for Ig-FcR interactions in immunity to this human pathogen.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Lipid Signaling via Pkh1/2 Regulates Fungal CO 2 Sensing through the Kinase Sch9.

Adaptation to alternating CO2 concentrations is crucial for all organisms. Carbonic anhydrases—metalloenzymes that have been found in all domains of life—enable fixation of scarce CO2 by accelerating its conversion to bicarbonate and ensure maintenance of cellular metabolism. In fungi and other eukaryotes, the carbonic anhydrase Nce103 has been shown to be essential for growth in air (~0.04% CO2). Expression of NCE103 is regulated in response to CO2 availability. In Saccharomyces cerevisiae, NCE103 is activated by the transcription factor ScCst6, and in Candida albicans and Candida glabrata, it is activated by its homologues CaRca1 and CgRca1, respectively. To identify the kinase controlling Cst6/Rca1, we screened an S. cerevisiae kinase/phosphatase mutant library for the ability to regulate NCE103 in a CO2-dependent manner. We identified ScSch9 as a potential ScCst6-specific kinase, as the sch9? mutant strain showed deregulated NCE103 expression on the RNA and protein levels. Immunoprecipitation revealed the binding capabilities of both proteins, and detection of ScCst6 phosphorylation by ScSch9 in vitro confirmed Sch9 as the Cst6 kinase. We could show that CO2-dependent activation of Sch9, which is part of a kinase cascade, is mediated by lipid/Pkh1/2 signaling but not TORC1. Finally, we tested conservation of the identified regulatory cascade in the pathogenic yeast species C. albicans and C. glabrata. Deletion of SCH9 homologues of both species impaired CO2-dependent regulation of NCE103 expression, which indicates a conservation of the CO2 adaptation mechanism among yeasts. Thus, Sch9 is a Cst6/Rca1 kinase that links CO2 adaptation to lipid signaling via Pkh1/2 in fungi

Differential cross section measurements for the production of a W boson in association with jets in proton–proton collisions at √s = 7 TeV

Measurements are reported of differential cross sections for the production of a W boson, which decays into a muon and a neutrino, in association with jets, as a function of several variables, including the transverse momenta (pT) and pseudorapidities of the four leading jets, the scalar sum of jet transverse momenta (HT), and the difference in azimuthal angle between the directions of each jet and the muon. The data sample of pp collisions at a centre-of-mass energy of 7 TeV was collected with the CMS detector at the LHC and corresponds to an integrated luminosity of 5.0 fb[superscript −1]. The measured cross sections are compared to predictions from Monte Carlo generators, MadGraph + pythia and sherpa, and to next-to-leading-order calculations from BlackHat + sherpa. The differential cross sections are found to be in agreement with the predictions, apart from the pT distributions of the leading jets at high pT values, the distributions of the HT at high-HT and low jet multiplicity, and the distribution of the difference in azimuthal angle between the leading jet and the muon at low values.United States. Dept. of EnergyNational Science Foundation (U.S.)Alfred P. Sloan Foundatio

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good