1,720,991 research outputs found
Regulation of the NKG2D stress ligands MICA, MICB and ULBP2 in proliferating and senescent tumor cells
No full textDas maligne Melanom stellt die tödlichste Form aller Hautkrebserkrankungen dar und entsteht durch eine maligne Transformation der Melanozyten. Melanomzellen können durch
zytotoxische Lymphozyten, wie NK Zellen und T Zellen als entartet erkannt und getötet werden. Beide Lymphozytensubgruppen kennzeichnet die Expression des Rezeptors NGK2D, der Stress-induzierte Liganden der MIC und ULBP Molekülfamilie auf den
Tumorzellen detektiert und damit zur Aktivierung der mmuneffektoren beiträgt.
Melanomzellen exprimieren die NKG2D Liganden (NKG2DL) MICA, MICB und ULBP2 auf ihrer Oberfläche. Die Rolle onkogener Signalwege in der Regulation der NKG2DL Expression in Melanomzellen ist bislang ungeklärt. Neben einer konstitutiven Aktivierung des MAP-Kinase Signalwegs, gesteuert durch die BRAF Mutante V600E, ist die PI3K-AKT
Signalkaskade wichtig für die Proliferation und das Überleben der Melanomzellen. Da die Hemmung der PI3K in einer Repression der Proliferation mündet, wird diese Kinase als Wirkstoffziel für die Tumortherapie untersucht. Die Proliferationsblockade kann von
transienter oder permanenter Natur sein - letzteres, einhergehend mit der Ausbildung eines seneszenten Phänotyps der Tumorzellen.
Im Rahmen der vorliegenden Arbeit sollte untersucht werden, inwiefern PI3K- sowie AKTabhängige Signale die NKG2DL Expression in Melanomzellen beeinflussen und wie sich Seneszenz auf die NKG2DL Expression auswirkt. Dazu wurde zunächst die Aktivität der
PI3K in verschiedenen Melanomzelllinien durch den Inhibitor LY294002 gehemmt. Dies führte zu einer verminderten MICA, MICB und ULBP2 Oberflächenexpression, unabhängig von deren BRAF Mutationsstatus. Eine stärkere Herabregulation konnte durch Verwendung des klinisch relevanten Inhibitors GSK2126458 erzielt werden, der in deutlich niedrigerer Konzentration zu einer Blockade der PI3K Signalgebung führte, gemessen anhand der Phosphorylierung der PI3K-kontrollierten Kinase AKT sowie stromabwärts gelegener
Zielstrukturen des Signalweges. Die PI3K Blockade verursachte neben einer verminderten Oberflächenexpression eine deutliche Reduktion des NKG2DL Gesamtproteins und der spezifischen mRNA, was bereits vier Stunden nach Inhibitorbehandlung detektierbar war.
Die Herabregulation der NKG2DL Oberflächenexpression durch den Inhibitor GSK2126458 resultierte in einer verringerten NKG2D-abhängigen NK Zellerkennung der Melanomzellen.
Da bekannt ist, dass eine Blockade der PI3K Signalgebung indirekt mit einer Aktivierung des Tumorsuppressors p53 einhergeht, wurde die Rolle von p53 unter Verwendung der
syngenen Kolonkarzinomzelllinien HCT116p53+/+ und HCT116p53-/- überprüft. In beiden Zelllinien führte die PI3K Inhibition zu einer Reduktion der MICA, MICB und ULBP2
Expression, wenngleich diese in den HCT116p53+/+ Zellen stärker ausfiel. In jedem Fall konnte eine NKG2DL Herabregulation auf der Ebene des Oberflächen- und Gesamt-Proteins sowie der mRNA beobachtet werden. Um zu prüfen, inwieweit AKT in der Regulation der NKG2DL Expression involviert ist, wurden Melanomzellen mit dem AKT Inhibitor GSK2110183 behandelt. Die Inhibitorexposition verursachte eine schwächere ULBP2 Expression auf der Oberfläche, auf Gesamtprotein- sowie mRNA-Ebene, während die Effekte auf die MICA und MICB Expression nicht konsistent ausfielen. Eine Herabregulation von ULBP2 konnte unter AKT Inhibition auch in HCT116p53+/+ und HCT116p53-/- Zellen beobachtet werden.
Dementsprechend führte die transiente Transfektion von Melanomzellen mit myristoyliertem und damit konstitutiv aktivem AKT1 (myrAKT1) zur Hochregulation der ULBP2
Proteinexpression. Dies konnte ebenfalls in Melanomzellen detektiert werden, die nach Transduktion myrAKT3 stabil exprimierten. Diese Daten demonstrieren, dass die Aktivität PI3K-abhängiger Signaltransduktionswege das NKG2DL Expressionsniveau bestimmt,
wobei die Regulation der ULBP2 Expression zudem auch AKT-abhängig erfolgt. Dies verdeutlicht, dass MICA, MICB und ULBP2 durch PI3K- und AKT-abhängige Wege differentiell reguliert werden.
Um gezielt die Auswirkung von Seneszenz auf die NKG2DL Expression zu ermitteln, wurde ein induzierbares Modell basierend auf der Melanomzelllinie WMM1175_p16INK4a verwendet.
In diesen Zellen wird Seneszenz über die IPTG-induzierbare Expression von p16 initiiert. Der irreversible Proliferationsarrest der malignen Zellen wurde über die verstärkte Expression von Seneszenz-assoziierter β-Galaktosidase, einer Hochregulation von p16, Herabregulation von E2F-1 und Ki-67 sowie Zunahme von Autofluoreszenz verifiziert. Interessanterweise
zeigten seneszente WMM1175_p16INK4a Zellen im Vergleich zu proliferierenden Kontrollzellen eine Induktion der NKG2DL MICA und MICB, während die ULBP2 Expression unbeeinflusst blieb. Im Gegensatz dazu führte replikative Seneszenz, induziert in Fibroblasten durch stetige Kultivierung, lediglich zu einer geringen Induktion der MICA und MICB Proteinexpression und einer leichten Herabregulation von ULBP2.
Zusammenfassend demonstrieren die Resultate dieser Doktorarbeit, dass die Expression von MICA, MICB und ULBP2 sowohl im Melanom, als auch im Kolonkarzinom durch PI3Kabhängige Signaltransduktionswege reguliert wird. Entsprechend führt die PI3K Blockade zur Herabregulation der NKG2DL Expression, einhergehend mit einem Proliferationsstopp.
Letzteres ist jedoch nicht zwangsläufig mit einer verminderten NKG2DL Expression assoziiert, da seneszente Melanomzellen eine Hochregulation von MICA und MICB aufweisen. Daraus lässt sich schlussfolgern, dass sowohl die PI3K-vermittelte onkogene
Proliferation als auch irreversibler Zellzyklusarrest im Kontext einer malignen Zelle, d.h. die Existenz „entarteter“ Zellen, dem Immunsystem via NKG2DL signalisiert werden.Melanoma is the most dangerous type of skin cancer, originating from malignant transformation of melanocytes. Melanoma cells can be detected as abnormal self and eliminated by cytotoxic lymphocytes of the innate and adaptive immune system such as NK and T cells. Both lymphocyte subsets express the activating receptor NKG2D. Its stress
inducible ligands, members of the MIC and ULBP molecule family, are expressed on tumor cells and signaling via NKG2D enhances the cytotoxic anti-tumor activity of T cells and NK cells. Though expression of the NKG2D ligands (NKG2DL) MICA, MICB and ULBP2 on melanoma cells has been demonstrated, their regulation by oncogenic signaling pathways remains to be elucidated. Melanoma cells are addicted to constitutive MAP-kinase pathway activation, frequently driven by mutant BRAFV600E and to oncogenic PI3K-AKT signaling both
contributing to cell proliferation and survival. As PI3K inhibition effectively blocks proliferation of melanoma cells it is considered an interesting drug target for tumor therapy. The cell cycle
arrest induced upon PI3K inhibition can either be transient or permanent, the latter associated with a senescent phenotype. Thus, the aim of this thesis was, at first, to analyse the impact of oncogenic PI3K and AKT signaling on NKG2DL expression and, at second, to
determine the influence of senescence on NKG2DL expression.
To assess whether PI3K dependent signaling pathways are involved in the regulation of NKG2DL expression in melanoma cells, different melanoma cell lines were treated with the PI3K inhibitor LY294002. This led to a decrease of MICA, MICB and ULBP2 surface expression independent of mutant BRAFV600E activity. NKG2DL downregulation was much stronger in the presence of the clinically relevant PI3K inhibitor GSK2126458. Compared to LY294002, blockade of PI3K dependent signaling by GSK2126458 was much more efficient
even at lower concentrations as determined by the phosphorylation level of PI3K downstream targets such as AKT. Reduced NKG2DL expression was detectable within 4 hours after PI3K inhibition at the levels of surface protein, total protein and mRNA. Notably,
inhibition of PI3K signaling decreased the NK cell sensitivity of melanoma cells. PI3K pathway inhibition has been demonstrated also to the induce activity of the tumor suppressor p53 indirectly via AKT. To study the role of p53 the PI3K-dependent regulation of
NKG2DL expression, the syngenic HCT116 p53+/+ and HCT116 p53-/- colon carcinoma cell system was used. Both cell lines showed diminished cell surface, total protein and mRNA Expression of MICA, MICB and ULBP2 under PI3K blockade, though effects were stronger in
the p53+/+ cell line. To analyse the role of AKT in regulating NKG2DL abundance, melanoma cells were exposed to the AKT Inhibitor GSK2110183. While blockade of AKT signaling had no consistent effects
on MICA and MICB expression it consistently diminished ULBP2 expression at the cell surface, at the levels of total protein and mRNA. Also HCT116p53+/+ and HCT116p53-/- cells responded to AKT inhibition with a decrease in ULBP2 expression. Accordingly, transient transfection of melanoma cells with myristoylated AKT1 (myrAKT1), rendering AKT constitutively active, increased ULBP2 protein expression. These results were confirmed in myrAKT3 stably transduced melanoma cells. These data demonstrate that the activity of PI3K dependent signaling determines the NKG2DL expression levels. As only ULBP2
expression is consistently affected by AKT dependent pathways this points to a differential regulation of MICA, MICB and ULBP2 by PI3K and AKT dependent signaling.
To assess the impact of senescence on NKG2DL expression the melanoma cell line WMM1175_p16INK4a was used as a model. In this cell line senescence via induction of p16 expression is inducible by IPTG. The irreversible cell cycle arrest was demonstrated by
increased expression of senescence-associated β-galactosidase and p16, decreased E2F-1 and Ki-67 protein levels and elevated autofluorescence. Interestingly, senescence of WMM1175_p16INK4a cells was accompanied by an increase in the expression of MICA and
MICB, while ULBP2 abundance was not affected. In contrast, replicative fibroblasts showed a slight increase in MICA as well as MICB and low reduction of ULBP2 expression.
In sum the data of this PhD thesis demonstrate that expression of MICA, MICB and ULBP2 is regulated by PI3K dependent pathways, in melanoma and colon carcinoma. Accordingly,
PI3K inhibition leads to downregulation of NKG2DL expression associated with a block in proliferation. The latter is not necessarily correlated with a diminished NKG2DL expression
as senescent melanoma cells upregulate MICA and MICB expression. Thus not only oncogenic proliferation but also irreversible cell cycle arrest of malignant cells, i.e. the existence of “abnormal“ cells, is signalled via NKG2DL to the immune system
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Die Modulation des Ubiquitin-Proteasom-Systems als Immunevasionsmechanismus des malignen Melanoms
Get PDFDie effiziente Präsentation von Tumorepitopen stellt einen kritischen Faktor zur Eliminierung von Tumorzellen durch die CD8+ cytotoxische T-Lymphozyten (CTL) vermittelte Immunantwort dar. Eine wichtige Rolle spielt in diesem Zusammenhang die effiziente Generierung von Tumorepitopen durch das Ubiquitin-Proteasom-System (UPS). Veränderungen von Komponenten des UPS, die an der Degradation und Prozessierung von Antigenen beteiligt sind, können daher zur Immunevasion von Tumorzellen gegenüber CTL führen. In der vorliegenden Arbeit wurden zwei unterschiedliche UPS-assoziierte Immunevasionsmechanismen des malignen Melanoms identifiziert, die auf einer ineffizienten Präsentation des immundominanten Tumorepitops Melan-A26-35 basieren. Ein Mechanismus beruht auf den unterschiedlichen katalytischen Eigenschaften von Proteasomsubtypen, deren Expression durch INFgamma induziert wird. Proteasomen, die einerseits die Immunountereinheiten beta1i und/oder beta2i beinhalten, oder anderseits mit dem Proteasomaktivator 28 (PA28) assoziiert sind, führen zu einer drastisch reduzierten Generierung des Tumorepitops Melan-A26-35. In beiden Fällen ist dies auf eine ineffiziente Prozessierung des N-Terminus des Epitops zurückzuführen. Der andere Immunevasionsmechanismus steht im Zusammenhang mit der ER-assoziierten Degradation (ERAD), die den retrograden Transport von Proteinen aus dem Endoplasmatischen Retikulum (ER) ins Cytosol zur proteasomalen Degradation beinhaltet. Durch Immunselektion mittels Melan-A26-35-spezifischen CTL wurden cytolyseresistente Melanomzellen identifiziert, deren Resistenz auf eine defiziente ER-assoziierte Degradation zurückzuführen ist. Dieser Defekt beruht auf einer verminderten Expression von ERAD-Komponenten, deren Reduktion die Verfügbarkeit des Antigens Melan-A zur proteasomalen Degradation und Generierung des immundominanten Epitops Melan-A26-35 wesentlich limitiert.Efficient presentation of tumor epitopes by MHC class I molecules on the cell surface is a prerequisite for the elimination of tumor cells by cytotoxic CD8+ T lymphocytes. The generation of these epitopes requires the degradation and processing of proteins by the ubiquitin proteasome system (UPS). Therefore alterations of UPS components can lead to tumor escape from immune recognition as a result of decreased epitope generation. In the present thesis two different UPS connected immune escape mechanisms of melanoma cells were identified. Both are based on an impaired generation of the immunodominant epitope Melan-A26-35 derived from Melan-A/MART-1 tumor antigen. One mechanism is mediated by the expression of different INFgamma-inducible proteasome immunosubunits leading to the formation of intermediate proteasome subtypes, which differ in their cleavage site preferences. Purified proteasomes harboring the immunosubunits beta1i and/or beta2i show a dramatic decrease in the generation of the Melan-A26-35 epitope. In addition, the INFgamma induced association of proteasomes with the proteaosome activator 28 (PA28) results in a reduced epitope generation. Both mechanisms are induced by an inefficient processing of the epitope’s N-terminus. The second immune escape mechanism is caused by defects of the ER-associated degradation pathway (ERAD). ERAD mediates the transport of ER-proteins back to the cytosolic compartment for proteasomal degradation. Via immunselection of tumor cells with Melan-A26-35 specific CTL, cytolysis resistant cells were identified. Resistance to CTL mediated lysis was shown to be connected to a decreased expression of ERAD components. This defect of the ERAD pathway limits the availability of the Melan-A protein and as consequence the generation of the immunodominant Melan-A26-35 epitope by proteasomes
- …
