Die Untersuchung der posttranskriptionellen Genregulation erfordert eine eingehende Kenntnis vieler molekularer Prozesse, die auf RNA wirken, von der Prozessierung im Nukleus bis zur Translation und der Degradation im Zytoplasma. Mit dem Aufkommen von RNA-seq-Technologien können wir nun jeden dieser Schritte mit hohem Durchsatz und Auflösung verfolgen. Ribosome Profiling (Ribo-seq) ist eine RNA-seq-Technik, die darauf abzielt, die präzise Position von Millionen translatierender Ribosomen zu detektieren, was sich als ein wesentliches Instrument für die Untersuchung der Genregulation erweist. Allerdings ist die Interpretation von Ribo-seq-Profilen über das Transkriptom aufgrund der verrauschten Daten und unserer unvollständigen Kenntnis des translatierten Transkriptoms eine Herausforderung. In dieser Arbeit präsentiere ich eine Methode, um translatierte Regionen in Ribo-seq-Daten zu erkennen, wobei ein Spektralanalyse verwendet wird, die darauf abzielt, die ribosomale Translokation über die übersetzten Regionen zu erkennen. Die hohe Sensibilität und Spezifität unseres Ansatzes ermöglichten es uns, eine umfassende Darstellung der Translation über das menschlichen und pflanzlichen (Arabidopsis thaliana) Transkriptom zu zeichnen und die Anwesenheit bekannter und neu-identifizierter translatierter Regionen aufzudecken. Evolutionäre Konservierungsanalysen zusammen mit Hinweisen auf Proteinebene lieferten Einblicke in ihre Funktionen, von der Synthese von bisher unbekannter Proteinen einerseits, zu möglichen regulatorischen Rollen andererseits. Darüber hinaus zeigte die Quantifizierung des Ribo-seq-Signals über annotierte Genemodelle die Translation mehrerer Transkripte pro Gen, was die Verbindung zwischen Translations- und RNA-Überwachungsmechanismen offenbarte. Zusammen mit einem Vergleich verschiedener Ribo-seq-Datensätze in menschlichen und planzlichen Zellen umfasst diese Arbeit eine Reihe von Analysestrategien für Ribo-seq-Daten als Fenster in die vielfältigen Funktionen des exprimierten Transkriptoms. / The study of post-transcriptional gene regulation requires in-depth knowledge of multiple molecular processes acting on RNA, from its nuclear processing to translation and decay in the cytoplasm. With the advent of RNA-seq technologies we can now follow each of these steps with high throughput and resolution. Ribosome profiling (Ribo-seq) is a popular RNA-seq technique, which aims at monitoring the precise positions of millions of translating ribosomes, proving to be an essential tool in studying gene regulation. However, the interpretation of Ribo-seq profiles over the transcriptome is challenging, due to noisy data and to our incomplete knowledge of the translated transcriptome. In this Thesis, I present a strategy to detect translated regions from Ribo-seq data, using a spectral analysis approach aimed at detecting ribosomal translocation over the translated regions. The high sensitivity and specificity of our approach enabled us to draw a comprehensive map of translation over the human and Arabidopsis thaliana transcriptomes, uncovering the presence of known and novel translated regions. Evolutionary conservation analysis, together with large-scale proteomics evidence, provided insights on their functions, between the synthesis of previously unknown proteins to other possible regulatory roles. Moreover, quantification of Ribo-seq signal over annotated transcript structures exposed translation of multiple transcripts per gene, revealing the link between translation and RNA-surveillance mechanisms. Together with a comparison of different Ribo-seq datasets in human cells and in Arabidopsis thaliana, this work comprises a set of analysis strategies for Ribo-seq data, as a window into the manifold functions of the expressed transcriptome.

Das CCAAT/Enhancer-Binding-Protein α (C/EBPα) ist ein Transkriptionsfaktor, der das Zellschicksal des hämatopoetischen Systems bestimmt. C/EBPα reguliert die Selbsterneuerung hämatopoetischer Stammzellen und bestimmt die myelomonozytäre Zelldifferenzierung. C/EBPα besitzt zudem die Eigenschaft lymphoide Zellen in myeloischen Zellen zu transdifferenzieren. Die von C/EBPα induzierte lymphoid-myeloische Transdifferenzierung kann als Modellsystem dienen, um die Vorgänge der Linienfestlegung, der zellulären Plastizität sowie der Funktionen von C/EBPα in der Zelldifferenzierung und Leukämogenese zu untersuchen. C/EBPα ist ein unstrukturiertes Protein, das seine Funktionen durch wechselnde Proteininteraktionen ausübt. Intrinsisch unstrukturierte Proteine, wie C/EBPα, sind prädestiniert an schwachen, multivalenten und hochdynamischen Proteininteraktionen teilzunehmen. Solche Inter-aktionen werden hauptsächlich durch kurze lineare Motive der Protein-Primärstruktur vermittelt. Motiv-basierte Proteininteraktionen sind mit den herkömmlichen Methoden der Interaktom-Analyse schwer zu analysieren. Die Anwendung der neuartigen Biotin-Ligase basierten TurboID Methode mit schneller Enzym-Kinetik ermöglichte nun die Analyse eines dynamischen C/EBPα Interaktoms während der lymphoid-myeloiden Transdifferenzierung. Es wurde festgestellt, dass sich die Proteinexpression der meisten C/EBPα interagierenden Proteine während der Transdifferenzierung kaum änderte, trotz erheblicher Änderungen des C/EBPα Interaktoms, was eine Regulation durch alternative Mechanismen nahelegt. Es wurden mehrere epigenetische Komplexe gefunden, einschließlich Mediator, SWI/SNF und CAF-1, die als mögliche Verbindung zwischen Interaktom, Transkriptom, Chromatinstruktur und Phänotyp in Betracht gezogen werden können. / The CCAAT/enhancer binding protein α (C/EBPα) is a key lineage-instructive transcription factor in the haematopoietic system. C/EBPα regulates the self-renewal of haematopoietic stem cells and is one of the main determinants of myeloid commitment. C/EBPα induces transdifferentiation of B cells into myeloid cells. C/EBPα-induced lymphoid-myeloid transdifferentiation may serve as a model system to study lineage commitment and cellular plasticity as well as address open questions related to functions of C/EBPα in differentiation and leukaemogenesis. C/EBPα is an intrinsically disordered protein that exerts its functions through protein interactions. Intrinsically disordered proteins (IDPs) engage in highly dynamic, weak, and multivalent protein interactions, which are mediated by short linear motifs (SLiMs). SLiMs-based protein interactions are difficult to analyse by conventional methods of interactome analysis. However, to understand the connection between the C/EBPα interactome and its functions, analysis in the cellular context is required. The application of the novel biotin ligase TurboID with faster kinetics enabled the analysis of the dynamic interactome of C/EBPα in the process of lymphoid-myeloid transdifferentiation. For most of the interacting proteins, the protein level did not change, yet variable interactions were found, indicating regulated interactions. TurboID identified changes in the composition of the SWI/SNF complex during transdifferentiation, including the exchange of subunits specific for BAF and PBAF subcomplexes, Brg1 and Brm ATPases, and cell type-specific subunits. The analysis also identified the interaction pattern of the histone demethylase Kdm6b and functional assays confirmed its role in transdifferentiation. TurboID enabled the comparative analysis of the interactomes of C/EBPα isoforms and mutants, that alter the balance between differentiation and proliferation and its oncogenic functions.

Urinary bladder cancer is a heterogeneous disease appearing in different forms, e.g. non-muscle invasive and muscle invasive. For all variants, the expression of proteins is interesting to analyze for diagnostic, predictive, prognostic and drug targeting purposes, since it reflects the altered gene expression causing the cancer. Since urothelial cells of the bladder are in direct contact with urine it is likely that this body fluid contains cancer-related proteins. In Paper I, unbiased analysis of proteins in urine from urinary bladder cancer patients and controls, using label-free quantification by mass spectrometry, was applied and four interesting proteins APOE, FGB, LRG and SERPINA1 were selected and further analyzed with western and dot blot. In Paper II, two more proteins, POLR1E and TOP2A, were validated as relevant proteins in bladder cancer urine. In Paper III and IV, the proteins GAL1 and STMN1 were investigated for their prognostic and therapeutic target potential in bladder cancer. In Paper II, III and IV, the expression of seven of the proteins were analyzed on tissue microarrays representing tumour tissue from 360 patients with different tumour stages. For the proteins identified by the urine screening approach, their protein expressions were confirmed in bladder cancer tissue. The expression level in tissue of five of the proteins, APOE, FGB, POLR1E (Paper II), GAL1 (Paper III) and STMN1 (Paper IV), increased with tumour stage, showing diagnostic relevance and three of the proteins, SERPINA1 (Paper II), STMN1 (Paper IV) and GAL1 (Paper III) had prognostic potential in urinary bladder cancer. In addition, GAL1 and STMN1 were demonstrated to be highly expressed in metastatic disease and inhibition of STMN1 reduced cell growth (Paper III and IV), indicating that these proteins are promising drug targets in urinary bladder cancer. In conclusion, the approach of this thesis has generated several candidate protein biomarkers in urine and tissue, validated with independent methods, which have the potential to improve the care for bladder cancer patients.

No description available.

No description available.

Selenium (Se) is an essential micronutrient and is also used in various industrial processes. However, Se also exhibits a low toxicity threshold and therefore presents a significant risk to human kind when released into the environment. The gap between Se deficiency (< 40 µg•day−1) and acute Se poisoning (> 400 µg•day−1) for humans is rather narrow. In addition, detrimental effects to the health of humans and other biota can arise from radioactive Se isotopes. Namely, 79Se is of concern, as it is one of the fission products originating from nuclear power production. The toxicity of selenium not only depends on its concentration but also on its speciation. This of course applies to both stable and radioactive isotopes. Microorganisms play a key role in determining and altering the speciation of Se in the selenium geochemical cycle. The naturally released selenium oxyanions (selenite (SeIVO32−) and selenate (SeVIO42−)) can be microbially reduced to differently shaped biogenic elemental selenium (BioSe, Se(0)) nanomaterials - BioSe-Nanospheres and BioSe-Nanorods. Even after more than 30 years of elaborated research on selenium, the impact of the microbial biota on the shape change of these BioSe-Nanomaterials lacks a fundamental understanding. Furthermore, due to the various species of microorganisms having different metabolisms, a detailed investigation of representative organism is required to predict the fate of selenium in the environment and engineered systems. Thus, the motivation behind this Ph.D. work was to study the effect of selected microorganisms (based on their high resilience, application in wastewater treatment processes, and capability to reduce selenium oxyanions) on the properties and fate of the produced biogenic elemental selenium nanomaterials. Namely, this meant deciphering the role of selenium oxyanion reduction mechanism on the localisation (intracellular or extracellular) of the microbially produced biogenic elemental selenium nanoparticles. This understanding is important as the localisation defines the release of the selenium nanoparticles in the environment and hence its potential pathway into the food chain. Further, the role of the microorganisms (pure culture and mixed culture) on the composition and stability of the corona (organic layer) on the BioSe-Nanomaterials was studied as properties of the corona can affect the stability and hence the localization of the nanomaterials. Moreover, the effect of the microbial environment on the shape establishment and stability, as well as on the fate of the produced biogenic elemental selenium nanomaterials was also investigated. Eventually, the obtained results narrow the identified knowledge gap and improve the understanding of the fate of selenium in the environment. In the first part of this Ph.D. thesis, the bacterial strain Bacillus safensis JG-B5T was chosen to study the influence of microbes on the fate of Se in the environment due to its occurrence in uranium mining sites where selenium is also found. First, this bacterium has been analysed by genome sequencing and its genomic data were deposited at the NCBI database. With the obtained results, the bacterial strain was classified in the corresponding phylogenetic tree. Furthermore, this Ph.D. work revealed that B. safensis JG-B5T is an obligate aerobic microorganism with the ability to reduce SeO32− to elemental selenium (Se(0)) in the form of red BioSe-Nanospheres. A reduction of SeO42− has not been observed. Two-chamber reactor experiments revealed that direct contact between SeO32− and the bacterial cells was necessary to start the reduction. In addition, microscopic investigations identified changes in the bacterial cell morphologies induced by toxic stress effects of SeO32−. Only extracellular production of BioSe-Nanospheres was observed using STEM equipped with a HAADF detector. The produced BioSe-Nanospheres were characterized by Raman spectroscopy as being amorphous Se. Furthermore, a stabilizing corona containing proteins and EPS, which caps the BioSe-Nanospheres, has been identified by FT-IR spectroscopy. The detailed composition of this corona has been further studied using proteomics analysis. The combination of two-chamber reactor experiments, genome analysis and the identified corona proteins indicated that the selenite reduction process of B. safensis JG-B5T was primarily mediated through membrane-associated proteins, like succinate dehydrogenase. Thus, a detailed molecular mechanism of the microbial reduction of SeO32− to BioSe-Nanospheres by the bacterial strain B. safensis JG-B5T has been proposed within this work. Besides these investigations on the formation of BioSe-Nanospheres, ζ-potential measurements have shown a low colloidal stability of the produced BioSe-Nanospheres. Thus, B. safensis JG-B5T is an attractive candidate in selenite wastewater treatment as it provides easy ways of recovering Se while maintaining low Se discharge. These investigations motivated us to study the general role of the microbial origin and microbial environment of the discharged nanomaterials in their shape change from BioSe-Nanospheres to BioSe-Nanorods. This constitutes the second part of this Ph.D. thesis. Thus, two different known microbial BioSe-Nanospheres producers by means of selenite reduction were used, namely the bacterial strain Escherichia coli K-12 and the microbial mix culture of anaerobic granular sludge. It was shown with Raman spectroscopy and SEM imaging that the BioSe-Nanospheres produced by E. coli K-12 remain amorphous and spherical when exposed to thermophilic conditions (up to one year), whereas those obtained by anaerobic granular sludge transform to trigonal BioSe-Nanorods. ζ-potential measurements identified a decrease of the colloidal stability of the transformed BioSe-Nanorods of anaerobic granular sludge compared to the still spherical BioSe-Nanospheres of E. coli K-12. As the shape of these BioSe-Nanospheres is stabilized by their corona, detailed investigations were performed to derive key factors affecting its shape change. CheSeNMs capped with different amount of BSA were produced and incubated to evaluate the quantitative effect of the amount of proteins in the corona on the shape stability of BioSe-Nanomaterials. This experiment implied that the larger quantity of proteins present in the corona of the BioSe-Nanospheres provide better shape stability. Indeed, the BioSe-Nanospheres produced by E. coli K-12 have 5.5 times more protein than those produced by anaerobic granular sludge. To gain deeper insight into their structural properties, proteomics analysis identified the surface proteins of the BioSe-Nanomaterials. The proteomics analysis also showed that the corona of BioSe-Nanospheres produced by E. coli K-12 consists of 1009 different proteins compared to only 173 on those produced by anaerobic granular sludge. The possible difference in the interaction of the corona proteins and selenium was elucidated using density functional theory calculations. The calculations suggest the possibility of the S-Se bond formation between Se atom and sulphur of the cysteine and methionine residues of the corona proteins. Furthermore, as representative for the microbial environment the bacterial strain B. safensis JG-B5T was used to mimic the role of microorganisms living in the vicinity of the discharged nanoparticles. The bacterial strain was incubated with purified BioSe-Nanospheres produced by E. coli K-12 at mesophilic conditions. Raman spectroscopy and SEM imaging showed that in contrast to the thermophilic incubation, the BioSe-Nanospheres transformed to BioSe-Nanorods in the presence of B. safensis JG-B5T. Proteomics analysis identified that the protein corona of BioSe-Nanospheres produced by E. coli K-12 was degraded by extracellular peptidases secreted upon co-incubation with B. safensis JG-B5T bacteria, which led to their transformation to BioSe-Nanorods. All the above findings show, how microorganisms fundamentally impact the speciation, colloidal stability, and shape of selenium. These, consequently, affect their flow coefficients or partition factors in the environment and therefore their fate. This work consequently demonstrates that the shape of the BioSe-Nanomaterials depends on both, their microbial origin and their microbial surrounding. Especially, the dynamic changes induced by this microbial environment on the shape of already formed BioSe-Nanospheres after their discharge are to be further explored. This increases the complexity in determining the risk assessment of Se and probably other redox active elements, which needs to be re-evaluated and improved by including microbial criteria for better accuracy. Based on the presented investigations, further studies regarding the detailed application and expansion to other bacterial strains will continuously widen the understanding of the behaviour of Se in the environment and engineered systems.

Alzheimers sjukdom (AD, eng. Alzheimer’s disease) upptäcktes för 111 år sedan av Alois Alz-heimer. Idag är det den ledande orsaken till demens hos äldre, och incidencen förväntas öka med befolkningens ökande livslängd. År 2050 förutspås antalet patienter med AD nå 10 miljoner personer [1]. Det har gjorts många försök att angripa AD via dess främsta kännetäcken, såsom plack av beta-amyloid (Aβ), Aβ-oligomerer, och ansamlingar av tau-protein, kallat tau-trassel. Trots att forskning om AD bedrivits i flera årtionden är dess orsak alltjämt okänd.På sistone har det funnits ett fokus på de inflammatoriska komponenterna inom AD. Det finns en utbredd aktivering av immunförsvaret i det centrala nervsystemet hos patienter med AD, men varken dess orsak eller dess roll inom AD är känd. Däremot finns det tydliga tecken på att inflammationen är av autoimmun art. Med detta i åtanke är det tydligt att det finns ett stort behov att utröna auto-immunitetens roll inom AD. I denna forskningsstudie användes proteomik-metoder för att bestämma autoantikroppsprofilerna inom plasma och cerebrospinalvätska (CSF, eng. cerebrospinal fluid) hos AD-patienter och en frisk kontrollgrupp.I denna studie användes par av plasma- och CSF-prover från 23 friska individer och 49 patien-ter. Dessutom inkluderades 2 plasmaprover och 18 CSF-prover från patienter. En 380-faldig och en 314-faldig riktad analys gjordes med hjälp utav suspension bead array-teknologi (SBA). Varje SBA bestod av färgkodade, magnetiska mikrosfärer i suspension, med antigen immobiliserade på kulornas yta. Denna analysmetod användes för att undersöka autoantikropssprofilerna i alla prover. Resul-taten visade en ökad respons från autoantikroppar mot antigenen SLC17A6 (Solute Carrier Family 17 Member 6), MAP1A (Microtubule Associated Protein 1A), och MAP2 (Microtubule Associated Protein 2) i patiener gentemot friska individer. Dock har dessa antigen uppvisat en bred reaktivitet i tidigare, opublicerade studier. Därför behövs ytterligare forskning för att fastställa deras roll inom AD.Dessutom användes paren av plasma- och CSF-prover för att undersöka autoantikroppsprofilernas överrensstämmelse inom varje patient. Det visade sig att korrelationen följde en normalfördelning, med starkare korrelation inom antigen med starkare reaktivitet mot den motsvarande autoantikroppen. Denna studie utgör en av de första storskaliga forskningsstudierna av överrensstämmelsen mellan autoantikroppsprofilerna inom plasma och CSF. / Alzheimer’s disease (AD) was discovered 111 years ago by Alois Alzheimer. Today, it is the leading cause of dementia in elderly, and incidence is expected to increase with life expectancy. By 2050, the number of a˙ected individuals is predicted to reach 10 million [1]. There have been numerous attempts to describe AD by its primary hallmarks, including amyloid plaques, amyloid beta (Aβ) oligomers, and tau tangles. However, despite several decades of intense research, the cause of AD remains unknown.Recently, there has been a focus on the inflammatory components of AD. There is an extensive activation of the immune system within the CNS of AD patients, but neither its cause nor its role in AD is known. However, there are strong indications that the inflammation has an autoimmune character. Considering this, there is an imperative need to examine autoimmunity within AD. In the present study, a proteomic approach was used to determine the autoantibody profiles within plasma and cerebrospinal fluid (CSF) within AD patients and healthy controls.Paired plasma and CSF samples from 23 healthy controls and 49 patients were included in the present study. In addition, 2 plasma samples and 18 CSF samples from patients were included (not paired). One 380-plex and one 314-plex targeted suspension bead array (SBA), each consisting of color-coded magnetic microspheres with immobilized antigens, were used to analyze autoantibody profiles in all samples. The resulting data revealed an increased autoantibody response towards anti-gens SLC17A6 (Solute Carrier Family 17 Member 6), MAP1A (Microtubule Associated Protein 1A), and MAP2 (Microtubule Associated Protein 2) in patients compared to healthy controls. However, as these antigens have displayed wide reactivities in previous, unpublished studies, they require further investigation to determine their role in AD.Furthermore, the paired CSF and plasma samples were used to investigate the correlation of autoantibody profiles within patients. The correlation was found to follow a normal distribution, with correlation being higher in antigens displaying stronger autoantibody reactivity. This work represents one of the first large-scale studies on the correlation of autoantibody profiles in plasma and CSF.

To investigate the influence of the internal micellar structure on the course of enzymatic cross-linking especially in the initial phase of the reaction, casein micelles isolated from raw milk via ultracentrifugation were incubated with microbial transglutaminase (mTG) in comparison with non-micellar sodium caseinate. Reactive lysine and glutamine residues were identified using a label-free approach, based on the identification of isopeptides within tryptic hydrolysates by targeted HRMS as well as manual monitoring of fragmentation spectra. Identified reactive sites were furthermore weighted by tracking the formation of isopeptides over an incubation time of 15, 30, 45 and 60 min, respectively. Fifteen isopeptides formed in the early stage of mTG cross-linking of caseins were identified and further specified concerning the position of lysine and glutamine residues involved in the reaction. The results revealed lysine K176 and glutamine Q175 of β-casein as the most reactive residues, which might be located in a highly flexible region of the molecule based on different possible reaction partners identified in this study. Except for the isopeptide αₛ₁ K34–αₛ₂ Q101 in sodium caseinate (SC), all reactive sites were detected in micellar and in non-micellar casein, indicating that the initial phase of enzymatic cross-linking is not affected by micellar aggregation of caseins.

In C. elegans bietet die frühe Embryogenese ein attraktives Modellsystem, um Wechselwirkungen von Proteinen in vivo zu entschlüsseln. Zur präzisen Identifizierung von spezifischen Interaktionen im C. elegans Embryo wurde ein neuer quantitativer Ansatz entwickelt, welcher die Expression von Fusionsproteinen an grün fluoreszierendes Protein in vivo mit markierungsfreier Interaktionsproteomik kombiniert. Diese Strategie wurde angewandt, um die Interaktionspartner von acht Proteinen zu untersuchen, die in essentiellen biologischen Prozessen während der frühen Embryogenese involviert sind. Diese Studie liefert als Ergebnis ein erstes embryonales in vivo Interaktionsnetzwerk bestehend aus 559 Interaktionen zwischen 472 Proteinen. Dieses Netzwerk erfasst nicht nur bekannte Bindungen, sondern auch neue Interaktionen von hoher funktioneller Relevanz. Die Netzwerkinformationen wurden mit Experimenten auf Basis der Ribonukleinsäuren-Interferenz kombiniert um neue Regulatoren der sogenannten „P granules” ausfindig zu machen. Infolgedessen wurde das fadenwurmspezifische Protein GEI-12 als neuer Interaktionspartner der DYRK-Kinase MBK-2 und als wichtiger Regler für die Dynamik der „P granules“ und für die Aufrechterhaltung der Keimbahn identifiziert. Dies führt zu einem hypothetischen Modell in welchem der Phosphorylierungszustand von GEI-12 den Auf- und Abbau der „P granules“ während der frühen Embryogenese vermittelt. Darüber hinaus veranlasst GEI-12 auch die Entstehung von „P granules“ in Säugetierzellen und bindet an PP2A-Phosphatasen, was darauf hindeutet, dass die grundlegenden biophysikalischen Eigenschaften die zur Entstehung der Ribonukleoprotein-Körperchen notwendig sind, im Laufe der Evolution zwischen Spezies konserviert geblieben sind. Zusammenfassend stellt die in vivo Interaktionskartierung ein vielseitiges Werkzeug dar, welches nicht nur die funktionelle Organisation des Proteoms aufdeckt, sondern auch Einsichten in die tierische Entwicklungsbiologie liefert. / In C. elegans, early embryogenesis provides an attractive model system for mapping in vivo protein interactions. In order to accurately identify specific interactions in C. elegans embryos, a new quantitative approach was developed combining in vivo expressed GFP fusion proteins with label-free interaction proteomics. This strategy was applied to studying the interaction partners of eight bait proteins involved in essential biological processes during early embryogenesis. As a result, this study generated a pilot embryo in vivo interaction network composed of 559 interactions among 472 proteins. Importantly, this network captures not only well-characterized bindings but also new interactions of high functional relevance. Further utility of the network is demonstrated by combining it with RNAi perturbation to search for new regulators of P granule formation in early embryos. Consequently, a worm-specific protein GEI-12 was discovered as a novel interaction partner of the DYRK kinase MBK-2 and as an important regulator of P granule dynamics and germline maintenance. This leads to a hypothetical model in which the phosphorylation state of GEI-12 mediates P granule assembly and disassembly during early embryogenesis. In addition, GEI-12 also induces granule formation in mammalian cells and interacts with PP2A phosphatases, indicating that the fundamental biophysical properties required for ribonucleoprotein granule formation are conserved across species during evolution. In summary, in vivo interactome mapping is a versatile approach that not only unravels the functional organization of the proteome but also can reveal insights into animal development.

Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde. / Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.