Functional analysis of the parvulin protein Par14

In der vorliegenden Arbeit sollten die Voraussetzungen für einen doppelten Knockout von Pin1 und Par14 innerhalb des DT40-Zellsystems geschaffen werden. Zunächst wurde das Zielkonstrukt für den Par14-Locus in einer mehrstufigen Klonierung generiert. Dies schloss 7 kb der genomischen Par14-Sequenz ein sowie eine LoxPErkennungssequenz innerhalb von Intron 1-2 sowie eine zweite LoxP-Stelle und einem Neomycin-Selektionsmarker innerhalb der 3‘-Sequenz. Mit diesem Konstrukt wurden DT40-MerCreMer-Zellen transfiziert und auf Insertion des Konstrukts selektiert. Der erfolgreiche Austausch des ersten Alles wurde mittels Southern Blot nachgewiesen, der hierfür im Labor erstmalig etabliert wurde. Der Austausch des zweiten Allels war auch nach mehrmaligenWiederholungen nicht erfolgreich. Das DT40-System mit stabil integrierter Cre-Rekombinase ermöglicht neben einem induzierbaren Knockout, das Ausschalten eines anderen Proteins (Doppel-Knockout). In der vorliegenden Arbeit konnte die genomische Sequenz des Pin1-Locus zwischen dem ersten und vierten Exon von Gallus gallus amplifiziert und sequenziert werden. Dies war nur nach methodischen Verbesserungen beim Amplifizieren und Klonieren von extrem GC-reichen Nukleotidsequenzen möglich. Somit liefert diese Arbeit die Voraussetzung für einen späteren Knockout des Pin1-Locus von Gallus gallus. Im Laufe der Arbeiten für den genetischen Knockout von Par14 in DT40-Zellen zeigte eine Studie von einer Arbeitsgruppe aus Japan eine Möglichkeit, die Expression von Par14 mittels der Knockdown-Methode zu reduzieren (Fujiyama-Nakamura et al., 2009). In der vorliegenden Arbeit konnte die Expression von Par14/17 mittels RNA-Interferenz durch die Verwendung von Stealth-siRNA-Konstrukten deutlich reduziert werden. Es konnte gezeigt werden, dass der Knockdown von Parvulin durch eine starke Änderung im Phänotyp sowohl beim Wachstum als auch im Zellzyklus-Profil begleitet wird. Als DNA-bindendes Protein könnte Par14 auch einen Einfluss auf das Transkriptom haben. Dieser Einfluss wurde durch Microarray-Hybridisierung nach einem siRNA-Knockdown untersucht. Hier zeigte Par14 keinen Effekt, der offensichtlich vergleichbar war mit den Microarrays von bekannten Transkriptionsfaktoren. Die Daten der Microarray-Analyse liefern jedoch Informationen für zukünftige Studien zu einer möglichen kompensatorischen Funktion von anderen Genen (z. B. für ribosomale Biogenese) bei Par14-Verlust.In the present work the conditions for a double knockout of Pin1 and Par14 within the DT40 cell line should be established. First the target construct for the Par14-locus was cloned in a multistep procedure. This encloses the 7 kb genomic sequence of Par14 as well as a LoxP-recognition sequence within Intron 1-2, the second LoxP place and a Neomycin-selection marker within 3' sequences. With this construct DT40-MerCreMer cells ware transfected and selected on insertion of the construct. The successful exchange of the first allele was confirmed by Southern Blot which was set up for this in the lab first. The exchange of the second allele was not successful. The DT40 system with stable expression of the Cre-Rekombinase allows both an inducible knockout and switching off another protein (double knockout). In the present work the genomic sequence of Pin1 locus between the first and the fourth exon of Gallus gallus was amplified and sequenced. This was possible only after methodical improvements of the amplification and cloning of the extreme GC rich nucleotide sequences. Therefore this work gives the possibility for a later knockout of the Pin1 locus of Gallus gallus. During this work for the genetic knockout of Par14 in DT40 cells a study of a working group from Japan showed, that it is possible to reduce the expression of Par14 by means of the knockdown method (Fujiyama-Nakamura et al., 2009). In the present work the expression of Par14 was clearly reduced by means of RNA interference technology with the stealth siRNA constructs. It could be shown that the knockdown of parvulin is associated with a strong change of the phenotype by the cell growth as well as the cell cycle profile. As a DNA-binding protein Par14 could also have an effect on the transkriptom. This effect was examined by microarray hybridisation. Here Par14 showed no effect which was obviously comparable with the microarrays of known transcription factors. Nevertheless, the data of the Microarray analysis deliver information for future studies to a possible compensatory function of other genes (e. g., for ribosomale biogenesis) by loss of Par14

Electronic band structure of a Tl/Sn atomic sandwich on Si(111)

A two-dimensional compound made of one monolayer of Tl and one monolayer of Sn on Si(111) has been found to have a sandwichlike structure in which the Sn layer (having the milk-stool arrangement) resides on the bulklike terminated Si(111) surface and the Tl layer (having the honeycomb-chained-trimer arrangement) is located above the Sn layer. The electronic band structure of the compound contains two spin-split surface-state bands, of which one is nonmetallic and the other is metallic. Near the Fermi level the metallic band is split with the momentum splitting Δk∥=0.037 Å−1 and energy splitting ΔEF=167 meV. The steep dispersion of the band when crossing the Fermi level corresponds to an electron velocity of ≈8.5×105 m/s, which is comparable to the value reported for graphene. The 2D Fermi contours have almost circular shape with spin texture typical for hexagonal surfaces

Single layer nickel disilicide on surface and as embedded layer

Single monolayers of various materials (e.g. graphene, silicene, bismuthene, plumbene, etc) have recently become fascinating and promising objects in modern condensed-matter physics and nanotechnology. However, growing a monolayer of non-layered material is still challenging. In the present report, it will be shown that single monolayer NiSi2 can be fabricated at Si(111) surface stabilized by either Tl, Pb or In monolayers. Nickel atoms were found to intercalate the stabilizing metal layers upon deposition and to reside in the interstitial sites inside the first silicon bilayer of bulk-like-terminated Si(111)1×1 surface. The interstitial positions almost coincide with the bulk NiSi2 atomic positions thus forming NiSi2 single layer. Atomic and electronic structure of formed systems is described in detail by means of a set of experimental techniques, including low-energy electron diffraction, scanning tunneling microscopy, angle-resolved photoemission spectroscopy and also first-principles density-functional-theory calculations. Quality of formed single monolayer NiSi2 was additionally confirmed by in situ four-probe transport measurements that show that single monolayer NiSi2 preserves a metallic-type conductivity down to 2.0 K. Moreover it was found that delta-type structure with atomic sheet of NiSi2 silicide embedded into a crystalline Si matrix can be fabricated using room-temperature overgrowth of a Si film onto the Tl stabilized NiSi2 surface layer. Confinement of the NiSi2 layer to a single atomic plane has been directly confirmed by high-resolution transmission electron microscopy

Synthesis of two-dimensional TlxBi1-x compounds and Archimedean encoding of their atomic structure

Crystalline atomic layers on solid surfaces are composed of a single building block, unit cell, that is copied and stacked together to form the entire two-dimensional crystal structure. However, it appears that this is not an unique possibility. We report here on synthesis and characterization of the one-atomic-layer-thick TlxBi1−x compounds which display quite a different arrangement. It represents a quasi-periodic tiling structures that are built by a set of tiling elements as building blocks. Though the layer is lacking strict periodicity, it shows up as an ideally-packed tiling of basic elements without any skips or halting. The two-dimensional TlxBi1−x compounds were formed by depositing Bi onto the Tl-covered Si(111) surface where Bi atoms substitute appropriate amount of Tl atoms. Atomic structure of each tiling element as well as arrangement of TlxBi1−x compounds were established in a detail. Electronic properties and spin texture of the selected compounds having periodic structures were characterized. The shown example demonstrates possibility for the formation of the exotic low-dimensional materials via unusual growth mechanisms

Human PAPS Synthase Isoforms Are Dynamically Regulated Enzymes with Access to Nucleus and Cytoplasm

In higher eukaryotes, PAPS synthases are the only enzymes producing the essential sulphate-donor 3′-phospho-adenosine-5′-phosphosulphate (PAPS). Recently, PAPS synthases have been associated with several genetic diseases and retroviral infection. To improve our understanding of their pathobiological functions, we analysed the intracellular localisation of the two human PAPS synthases, PAPSS1 and PAPSS2. For both enzymes, we observed pronounced heterogeneity in their subcellular localisation. PAPSS1 was predominantly nuclear, whereas PAPSS2 localised mainly within the cytoplasm. Treatment with the nuclear export inhibitor leptomycin B had little effect on their localisation. However, a mutagenesis screen revealed an Arg-Arg motif at the kinase interface exhibiting export activity. Notably, both isoforms contain a conserved N-terminal basic Lys-Lys-Xaa-Lys motif indispensable for their nuclear localisation. This nuclear localisation signal was more efficient in PAPSS1 than in PAPSS2. The activities of the identified localisation signals were confirmed by microinjection studies. Collectively, we describe unusual localisation signals of both PAPS synthase isoforms, mobile enzymes capable of executing their function in the cytoplasm as well as in the nucleus

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Atomic arrangement and electron band structure of Si(1 1 1)-ß-√3×√3-Bi reconstruction modified by alkali-metal adsorption: Ab initio study

Using ab initio calculations, atomic structure and electronic properties of Si(1 1 1)$sqrt{3} imes sqrt{3}$ -Bi surface modified by adsorption of 1/3 monolayer of alkali metals, Li, Na, K, Rb and Cs, have been explored. Upon adsorption of all metals, a similar atomic structure develops at the surface where twisted chained Bi trimers are arranged into a honeycomb network and alkali metal atoms occupy the ${{T}_{4}}$ sites in the center of each honeycomb unit. Among other structural characteristics, the greatest variation concerns the relative heights at which alkali metals reside with respect to Bi-trimer layer. Except for Li, the other metals reside higher than Bi layer and their heights increase with atomic number. All adsorbed surface structures display similar electron band structures of which the most essential feature is metallic surface-state band with a giant spin splitting. This electronic property allows one to consider the Si(1 1 1)$sqrt{3} imes sqrt{3}$ -Bi surfaces modified by alkali metal adsorption as a set of material systems showing promise for spintronic applications

Atomic arrangement and electron band structure of Si(1 1 1)-ß-√3×√3-Bi reconstruction modified by alkali-metal adsorption: Ab initio study

Using ab initio calculations, atomic structure and electronic properties of Si(1 1 1)$sqrt{3} imes sqrt{3}$ -Bi surface modified by adsorption of 1/3 monolayer of alkali metals, Li, Na, K, Rb and Cs, have been explored. Upon adsorption of all metals, a similar atomic structure develops at the surface where twisted chained Bi trimers are arranged into a honeycomb network and alkali metal atoms occupy the ${{T}_{4}}$ sites in the center of each honeycomb unit. Among other structural characteristics, the greatest variation concerns the relative heights at which alkali metals reside with respect to Bi-trimer layer. Except for Li, the other metals reside higher than Bi layer and their heights increase with atomic number. All adsorbed surface structures display similar electron band structures of which the most essential feature is metallic surface-state band with a giant spin splitting. This electronic property allows one to consider the Si(1 1 1)$sqrt{3} imes sqrt{3}$ -Bi surfaces modified by alkali metal adsorption as a set of material systems showing promise for spintronic applications