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Infection of most cell types with herpes simplex virus (HSV) mutants lacking the activation functions of VP16 and/or ICP0 results in repression of viral gene expression. However, the human osteosarcoma cell line U2OS supports the replication of VP16 and ICP0 mutants to nearly wild type levels. Prior to the studies presented in this thesis, the basis for the permissivity of U2OS cells to VP16 and ICP0 mutants had not been explored. Here, somatic cell fusion assays were used to determine that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate gene silencing mechanism. The artificial induction of interferon stimulated genes that occurs during the somatic cell fusion assays is not the basis for the observed repression of viral gene expression. As one means of identifying components of the antiviral pathway defective in U2OS cells, restrictive cell types were treated with kinase inhibitors and infected with VP16 and/or ICP0 mutants. Although several compounds were identified which compensate for the defect in gene expression of VP16 mutants, these drugs also stimulate mutant virus gene expression in U2OS. Thus, U2OS are most likely not defective in the cellular signalling pathway(s) targeted by these compound(s). Finally, the importance of VP16 and ICP0 in modulating chromatin structure on the viral genome in both restrictive and permissive cells was examined, uncovering an essential role for both proteins in altering histone occupancy and acetylation levels. Importantly, U2OS cells have a defect in the chromatin-based pathway targeted by ICP0. However, evidence suggests that the ability of VP16 and ICP0 to affect histone occupancy and acetylation levels is not required for viral gene expression. Taken together, the results of this thesis demonstrate that U2OS cells support the replication of VP16 and ICP0 mutants due to a defect in an innate antiviral mechanism which does not involve the targets of several well characterized kinase inhibitors. The significance of the defect in a chromatin-based pathway targeted by ICP0 in U2OS cells remains to be elucidated. / Virology

State-of-the-art fluorescent imaging research is strictly limited to eight fluorophore labels duringthe study of intercellular interactions among organelles. The number of excited fluorophore colorsis restricted due to overlap in the narrow spectra of visual wavelength. However, this requires aconsiderable effort of analysis to be able to tell the overlapping signals apart. Significant overlapalready occurs with the use of more than four fluorophores and is leaving researchers limited to asmall number of labels and the hard decision to prioritize between cellular labels to use. Except for the physical limitations of fluorescent labeling, the labeling itself causes behavioralabnormalities due to sample perturbation. In addition to this, the labeling dye or dye-adjacentantibodies are potentially causing phototoxicity and photobleaching thus limiting the timescale oflive cell imaging. Nontoxic imaging modalities such as transmitted-light microscopes, such asbright-field and phase contrast methods, are available but not nearly achieving images of thespecificity as when using fluorophore labeling. An approach that could increase the number of organelles simultaneously studied withfluorophore labels, while being cost-effective and nontoxic as transmitted-light microscopes wouldbe an invaluable tool in the quest to enhance knowledge of cellular studies of organelles. Here wepresent a deep learning solution, using convolutional neural networks built to predict thefluorophore labeling effect on the nucleus, from a transmitted-light input. This solution renders afluorescent channel available for another marker and would eliminate the process of labeling thenucleus with dye or dye-conjugated antibodies by instead using deep convolutional neuralnetworks. / Allra senaste forskningen inom fluorescensmikroskopi är begränsat till upp till åtta fluoroforer förstudier av intracellulära kommunikationer mellan organeller. Antalet fluorescerande färger ärbegränsade till följd av spektralt överlapp i det synliga våglängdsområdet. Överlappande signalerbehöver matematiskt bearbetas vilket innebär ökad arbetsinsats och signifikant överlappning skerredan vid användning av fler än fyra fluoroforer. Denna begräsning innebär i slutändan att forskarehar ett litet antal fluoroforer att arbeta med och behöver därmed prioritera vilka cellulära strukturersom kan märkas samtidigt. Utöver de spektrala begräsningarna med fluorescensmikroskopi, så innebär även själva färgningenav cellulära komponenter en negativ cellulär påverkan i form av avvikande beteende.Fluorescerande färgämnen och märkta antikroppar orsakar potentiellt fototoxicitet ochljusblekning, vilket begränsar tidsrymden vid studier av levande celler. Ljusfältsmikroskop sombright-field and faskontrast har inte en toxisk påverkan men producerar inte i närheten likadetaljerade bilder som fluorescensmikroskop gör. Ett tillvägagångssätt som skulle kunna öka antalet organeller som simultant kan undersökas medfluoroforer, som samtidigt är kostnadseffektiv och inte har en toxisk påverkan somljusfältsmikroskop, skulle vara ett ovärderligt verktyg för utökad kunskap vid cellulära studier avorganeller. Här presenteras en maskininlärningsmetod byggd med artificiella neuronnät för attpredicera fluorescerande infärgningen av cellkärnan i fluorescensmikroskop, med bilder frånljusfältsmikroskop. Denna lösning frigör en fluorofor som kan användas till andra organellersamtidigt som arbetet med fluorescerande infärgning av cellkärnan inte längre är nödvändigt ochersätts med ett artificiellt neuronnät.

Inositolpyrophosphate (PP InsPs) sind Signalmoleküle in eukaryotischen Zellen, die u.a. als Sensoren für ATP- und Phosphat fungieren, und insbesondere durch allosterische Regulation und posttranslationale Modifikationen (PTMs) wirken. Diese Arbeit ist in zwei Teile unterteilt, die sich auf zwei verschiedene ungewöhnliche Eigenschaften dieser Moleküle konzentrieren. Der erste Teil untersucht PP-InsPs in Lösung, mit Schwerpunkt auf ihrer Fähigkeit, abhängig von pH und Metallionen ihre Konformation zu ändern. Diese Eigenschaft ist einzigartig unter biologisch vorkommenden kleinen Molekülen. Drei eng verwandte Moleküle, InsP6, 5PP InsP5 und InsP8, wurden mittels NMR Spektroskopie charakterisiert, um herauszufinden, ob sie unter annähernd zytosolischen Bedingungen ihre Konformation ändern können. Dies war der Fall für InsP8, welches deshalb bezüglich Protonierung und Komplexbildung genauer untersucht wurde. Zu guter Letzt konnten ITC Experimente demonstrieren, dass eine Lösungsumgebung, die die Konformationsänderung von InsP8 begünstigt, auch seine Bindung an eine damit interagierende Proteindomäne verstärkt. Der zweite Teil beschäftigt sich mit der Pyrophosphorylierung von Proteinen, einer PTM, die nach derzeitigem Wissen non-enzymatisch von PP InsPs auf phosphorylierte Aminosäurereste übertragen wird – im Gegensatz zur enzymatischen Phosphorylierung durch Kinasen. Ein Probenvorbereitungsprotokoll zum Nachweis von endogener Pyrophosphorylierung in Zellen wurde entwickelt und mit synthetischen Standardpeptiden validiert. Anschließend wurde es an drei menschlichen Zelllinien erprobt, und konnte über einhundert Modifikationsstellen identifizieren, zumeist auf Proteinen im Zellkern. Dies beweist zum ersten Mal die Existenz von endogener Pyrophosphorylierung. Proteomics an Knockout-Zelllinien bestätigten die Hypothese, dass Pyrophosphorylierung von 5PP-InsP5 (InsP7) abhängig ist. Mikroskopie und qPCR-Experimente lieferten Hinweise auf eine Funktion in der Regulation der Ribosomenbiogenese. / Inositol pyrophosphates (PP-InsPs) are messenger molecules in eukaryotic cells, that serve as sensors of phosphate and ATP, among other functions, signaling e.g. through allosteric regulation and posttranslational modifications. This work is structured into two parts, focusing on two different unusual features of these molecules. The first part investigates PP-InsPs in solution, with emphasis on the messengers’ ability to undergo a pH and metal ion dependent conformational change, a feature unique among biological small molecules. Three closely related molecules, InsP6, 5PP InsP5 and InsP8 were characterized by NMR, to determine if they could change conformation under conditions approximating a cytosolic environment. This was the case for InsP8, which was therefore studied in more detail regarding protonation and complexation. Finally, ITC experiments showed that solution conditions favoring the conformational change of InsP8 also improved its binding to a known interacting protein domain. The second part of the thesis is concerned with protein pyrophosphorylation, a post-translational modification thought to be transferred non-enzymatically from PP InsPs to phosphorylated amino acid residues – opposed to the usual enzymatic phosphorylation through kinases. A sample preparation workflow for detection of endogenous pyrophosphorylation in cells has been developed and validated using synthetic standard peptides. It was then applied to three human cell lines, discovering more than one hundred modified sites, mostly on nuclear proteins, and proving for the first time the existence of endogenous pyrophosphorylation. Proteomics on knockout cell lines confirmed the hypothesis that pyrophosphorylation depends on 5PP-InsP5 (InsP7). Finally, microscopy and qPCR experiments suggested a regulatory role in ribosome biogenesis.