Anreicherung einer aktivitätsabhängigen Splice-Variante von Dynamin 1 in Synapsen in verschiedenen Regionen des Zentralen Nervensystems

Dynamine sind GTP-bindende Mechanoenzyme, die an verschiedenen Formen der Endozytose und Vesikelabschnürung beteiligt sind. Es sind drei Dynamin-Gene (Dynamin1- 3) bekannt. Dynamine sind mit verschiedenen Splicevarianten an sehr unterschiedlichen Vorgängen in der Zelle beteiligt. Diese umfassen sowohl konstitutive als auch aktivitätsabhängige Prozesse. Nervenzellen besitzen einen besonders aktiven endozytotischen Membranverkehr, insbesondere in den präsynaptischen Terminalen. In den präsynaptischen Terminalen geschieht synaptische Kommunikation via Exozytose von Neurotransmitter-enthaltenden synaptischen Vesikeln, die nach der Fusion mit der präsynaptischen Plasmamembran wieder aufgenommen werden müssen. Die endozytotische Wiederaufnahme von Membranvesikeln muss dabei dynamisch an unterschiedliche Aktivitätszustände in der präsynaptischen Terminale angepasst werden. In der vorliegenden Arbeit habe ich eine spezielle, aktivitätsabhängige Splicevariante (Dynamin 1xb) von Dynamin 1, Dynamin 1xb, untersucht. Die Dynamin1 Splicevariante Dynamin1xb ist besonders interessant, da sie eine Bindungsstelle für die Ca2+-abhängige Phosphatase Calcineurin besitzt (Bodmer et al., 2011). Die Ca2+- und damit aktivitätsgesteuerte Phosphatase Calcineurin dephosphoryliert Dynamin1 und kann auf diese Weise Dynamin1 „anschalten“ bzw. in seiner Aktivität steigern. Diese Dynamin1xb-abhängige Form der Endozytose kann während starker neuronaler Stimulation beim Recyceln von synaptischen Vesikeln von großer Bedeutung sein. Im Rahmen dessen habe ich einen selbst hergestellten monoklonalen Antikörper (Anti-Dynamin 1xb (Klon 1E10)) gegen diese Ca2+- regulierte, aktivitätsabhängige Splicevariante von Dynamin 1 charakterisiert. Mit Hilfe des hergestellten monoklonalen Antikörpers, der spezifisch Dynamin1xb erkennt, konnte ich Dynamin 1xb mittels Western Blot in verschiedenen zentralnervösen Geweben nachweisen: Cerebellum, Neocortex, Rückenmark und Retina. Dynamin1xb war dagegen in nicht neuronalem Gewebe, wie Niere und Darm, abwesend, wie ich mittels Western-Blot- Untersuchungen gezeigt habe. Die Spezifität des Antikörpers wurde mithilfe von Präabsorptionsexperimenten gezeigt. Die Präabsorption des Antikörpers mit dem spezifischen Dynaminpeptid, gegen welches der Antikörper gerichtet ist, führte sowohl in Western Blot-Untersuchungen als auch in Immunfluoreszenzexperimenten zu einem Verschwinden des Immunsignals. Um Dynamin 1xb genau lokalisieren zu können, verwendete ich den hergestellten, Dynamin1xb-spezifischen monoklonalen Antikörper (Anti-Dynamin1xb (Klon 1E10)) in der Immunfluoreszenzmikroskopie (konventionelle Epifluoreszenzmikroskopie und Konfokalmikroskopie) und untersuchte mit dieser Methode verschiedene Gewebe. Dabei zeigte sich, dass Dynamin 1xb im Cerebellum, im Rückenmark und im visuellen Cortex in der grauen Substanz angereichert ist, und zwar dort, wo sich die Synapsen befinden. Die weiße Substanz zeigte im Vergleich dazu ein nur sehr schwach ausgeprägtes Dynamin 1xb Immunsignal. Dynamin 1xb wies in Cerebellum und visuellem Cortex außerdem ein ähnliches Expressionsmuster wie die synaptischen Vesikelproteine SV2 und Synaptotagmin 1 sowie das aktive Zonen-Protein RIM1/2 auf. In der Retina wurde Dynamin 1xb in den Ribbonsynapsen der Photorezeptoren und in der Synapsenschicht zwischen den Bipolarund Ganglienzellen nachgewiesen, welches durch Doppelimmunfluoreszenzinkubationen mit einem Protein der Ribbonsynapsen, RIBEYE, bestätigt wurde. In Photorezeptor- Ribbonsynapsen war das Dynamin 1xb Signal besonders stark in der peri-aktiven Zone in unmittelbarer Nachbarschaft zu den synaptischen Ribbons. Zusammenfassend wurde Dynamin 1xb in allen synaptischen Schichten aller ausgetesteten Abschnitte des ZNS (cerebellären und visuellen Cortex, graue Substanz des Rückenmarks, OPL und IPL der Retina) gefunden und ist dort wahrscheinlich an der aktivitäts-abhängigen, Ca2+-regulierten Endozytose beteiligt. Eine Beteiligung von Dynamin 1xb an der aktivitätsabhängigen Endozytose in der präsynaptischen Terminale wird auch durch vergleichende semiquantitative Untersuchungen synaptischer Dynamin 1xb Immunsignale an hell- und dunkel-adaptierten Netzhäuten der Mausretina weiter gestützt.„The calcineurin-binding, activity-dependent splice variant dynamin 1xb is highly enriched in synapses in various regions of the central nervous system” Dynamins are GTP-binding mechanoenzymes involved in different types of endocytosis and vesicle scission. Three dynamin genes (dynamin 1-3) are known. Dynamins are with their different splice variants involved in different cellular processes. These include constitutive as well as activity-dependent processes. Neurons possess a particular active endocytotic membrane traffic, especially in the presynaptic terminals. In the presynaptic terminal synaptic communication via exocytosis of neurotransmitter-containing synaptic vesicles takes place. These vesicles need to be retrieved after fusion with the plasma membrane. Furthermore, endocytosis needs to be adopted to different levels of synaptic activity in the presynaptic terminal. In the present study, I examined a particular activity-dependent splice variant (dynamin 1xb) of dynamin 1. This dynamin 1xb splice variant is unique because it can directly interact with the calcium- and calmodulin-dependent phosphatase calcineurin (Bodmer, Ascaño, und Kuruvilla 2011). The calcium and therefore activity-dependent phosphatase calcineurin dephosphorylates dynamin 1 and is able to “turn on” dynamin 1 or to enhance its activity. This dynamin 1xb dependent endocytosis can have a strong impact on vesicle recycling during high levels of synaptic activity. For this purpose, I characterized a new mouse monoclonal antibody (anti-dynamin1xb (clone 1E10)) against this Ca2+- regulated, activity-dependent splice variant of dynamin 1. In parallel to localization analyses of dynamin 1xb in different neuronal tissue, I found in western blot experiments, that dynamin 1xb is highly expressed in cerebellum, neocortex, spinal cord, and retina. The antibody’s specificity was tested by performing pre-absorption experiments by incubating the dynamin peptide or a control peptide with the monoclonal antibody clone 1E10. Later on, pre-absorbed antibody was tested in western blot analyses and in immunofluorescence experiments where pre-absorbed antibody produced no signal. Furthermore specificity of antibody was confirmed by checking non-neuronal tissues like kidney and intestine where no immune reactivity was detected, when the dynamin 1xb antibody was preabsorbed with the dynamin peptide; while the immunoreactivity remained unchanged. To localize dynamin 1xb, I performed conventional epifluorescence microscopy and confocal microscopy. I found that dynamin 1xb is enriched in the grey matter of cerebellum, visual cortex and spinal cord, where synapses are localized. All three layers of the cerebellar cortex expressed dynamin 1xb. Markedly strong signals were detected in the cerebellar glomeruli of the granular layer. Likewise all layers of the visual cortex expressed dynamin 1xb. In spinal cord dynamin 1xb was highly enriched in the grey matter, equally distributed with synaptotagmin 1 and RIM 1/2, whereas tubulin was expressed in the grey and predominantly in the white matter. In contrast only a very weak dynamin 1xb immunosignal was detected in the fiber tracts of white matter of the cerebellum and spinal cord. Dynamin 1xb displayed a similar expression profile as the synaptic vesicle proteins SV2 and synaptotagmin 1 as well as the active zone protein RIM 1/2 in cerebellum and visual cortex. In retina dynamin 1xb was detectable in the outer and in the inner plexiform layer. In the photoreceptor ribbon synapses the dynamin 1xb signal was particularly strong in the periactive zone in close vicinity to the synaptic ribbons, proved by double immunofluorescence staining with RIBEYE. Taken together dynamin 1xb was found to be enriched in all layers of the cerebellar and the visual cortex, in the grey matter of the spinal cord, and in OPL and IPL of the retina playing a role in the activity-dependent, Ca2+-regulated endocytosis. An involvement of dynamin 1xb in activity-dependent endocytosis is further supported by semi quantitative evaluation of synaptic dynamin 1xb immunosignals between light- and darkadapted retinas

Characterization of glycine-N-acyltransferase like 1 (GLYATL1) in prostate cancer

BackgroundRecent microarray and sequencing studies of prostate cancer showed multiple molecular alterations during cancer progression. It is critical to evaluate these molecular changes to identify new biomarkers and targets. We performed analysis of glycine-N-acyltransferase like 1 (GLYATL1) expression in various stages of prostate cancer in this study and evaluated the regulation of GLYATL1 by androgen.MethodWe performed in silico analysis of cancer gene expression profiling and transcriptome sequencing to evaluate GLYATL1 expression in prostate cancer. Furthermore, we performed immunohistochemistry using specific GLYATL1 antibody using high-density prostate cancer tissue microarray containing primary and metastatic prostate cancer. We also tested the regulation of GLYATL1 expression by androgen and ETS transcription factor ETV1. In addition, we performed RNA-sequencing of GLYATL1 modulated prostate cancer cells to evaluate the gene expression and changes in molecular pathways.ResultsOur in silico analysis of cancer gene expression profiling and transcriptome sequencing we revealed an overexpression of GLYATL1 in primary prostate cancer. Confirming these findings by immunohistochemistry, we show that GLYATL1 is overexpressed in primary prostate cancer compared with metastatic prostate cancer and benign prostatic tissue. Low-grade cancers had higher GLYATL1 expression compared to high-grade prostate tumors. Our studies showed that GLYATL1 is upregulated upon androgen treatment in LNCaP prostate cancer cells which harbors ETV1 gene rearrangement. Furthermore, ETV1 knockdown in LNCaP cells showed downregulation of GLYATL1 suggesting potential regulation of GLYATL1 by ETS transcription factor ETV1. Transcriptome sequencing using the GLYATL1 knockdown prostate cancer cell lines LNCaP showed regulation of multiple metabolic pathways.ConclusionsIn summary, our study characterizes the expression of GLYATL1 in prostate cancer and explores the regulation of its regulation in prostate cancer showing role for androgen and ETS transcription factor ETV1. Future studies are needed to decipher the biological significance of these findings.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151252/1/pros23887.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151252/2/pros23887_am.pd

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Measurement of prompt open-charm production cross sections in proton-proton collisions at root s=13 TeV

The production cross sections for prompt open-charm mesons in proton-proton collisions at a center-of-mass energy of 13TeV are reported. The measurement is performed using a data sample collected by the CMS experiment corresponding to an integrated luminosity of 29 nb(-1). The differential production cross sections of the D*(+/-), D-+/-, and D-0 ((D) over bar (0)) mesons are presented in ranges of transverse momentum and pseudorapidity 4 < p(T) < 100 GeV and vertical bar eta vertical bar < 2.1, respectively. The results are compared to several theoretical calculations and to previous measurements.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

Measurement of B-c(2S)(+) and B-c*(2S)(+) cross section ratios in proton-proton collisions at root s=13 TeV

Peer reviewe

Measurement of the W gamma Production Cross Section in Proton-Proton Collisions at root s=13 TeV and Constraints on Effective Field Theory Coefficients

A fiducial cross section for W gamma production in proton-proton collisions is measured at a center-of-mass energy of 13 TeV in 137 fb(-1) of data collected using the CMS detector at the LHC. The W -> e nu and mu nu decay modes are used in a maximum-likelihood fit to the lepton-photon invariant mass distribution to extract the combined cross section. The measured cross section is compared with theoretical expectations at next-to-leading order in quantum chromodynamics. In addition, 95% confidence level intervals are reported for anomalous triple-gauge couplings within the framework of effective field theory.Peer reviewe

Development and validation of HERWIG 7 tunes from CMS underlying-event measurements

This paper presents new sets of parameters (“tunes”) for the underlying-event model of the HERWIG7 event generator. These parameters control the description of multiple-parton interactions (MPI) and colour reconnection in HERWIG7, and are obtained from a fit to minimum-bias data collected by the CMS experiment at s=0.9, 7, and 13Te. The tunes are based on the NNPDF 3.1 next-to-next-to-leading-order parton distribution function (PDF) set for the parton shower, and either a leading-order or next-to-next-to-leading-order PDF set for the simulation of MPI and the beam remnants. Predictions utilizing the tunes are produced for event shape observables in electron-positron collisions, and for minimum-bias, inclusive jet, top quark pair, and Z and W boson events in proton-proton collisions, and are compared with data. Each of the new tunes describes the data at a reasonable level, and the tunes using a leading-order PDF for the simulation of MPI provide the best description of the dat