Biologische Hochschulschriften (Goethe-Universität)
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Die im Rahmen dieser Arbeit durchgeführten Untersuchungen führten zu folgenden Ergebnissen: 1. Eindimensionale Gelelektrophoresen Die Analyse mitochondrialer Proteine aus juvenilen und seneszenten P. anserina-Wildstämmen mit Hilfe von eindimensionalen SDS- und eindimensionalen Blau-Nativen-Gelelektrophoresen zeigt keine deutlichen, seneszenzspezifischen Unterschiede. Im Gegensatz dazu werden in initialen Versuchen der nicht-radioaktiven 2D-PAGE differentiell gebildete Proteine visualisiert. 2. 2D-PAGE mit radioaktiv-markierten, mitochondrialen Proteinen aus jungen und alten P. anserina-Wildstämmen In der ungerichteten Proteomanalyse wurden 29 differentiell-gebildete Proteine identifiziert und zusätzlich zahlreiche Isoformen einiger Proteine gezeigt. Von der ß-ATPase wurden modifizierte Isoformen gefunden. Außerdem wurde eine seneszenspezifisch verringerte Bildung von ROS-Abwehr-Proteinen in den Mitochondrien detektiert. Im Gegensatz dazu wurde eine größere Menge eines Chaperons gefunden, das bei der Proteinsynthese eine Rolle spielt: eine Protein-Disulfid-Isomerase, die die Umlagerung und Neubildung von Di-Sulfid-Brücken bei der Faltung von Proteinen katalysiert. Zusätzlich wurde eine erhöhte Menge des Proteins SSC1 identifiziert. Dieses gehört zur Hsp70-Hitzeschock-Proteinfamilie. Es wurde ebenfalls eine erhöhte Menge des Apoptosefaktors Cyclophilin D in den mitochondrialen Proben aus den seneszenten Wildstämmen identifiziert. Die Identifizierung dieses Proteins in Mitochondrien von P. anserina stellt neben der Charakterisierung der Metacaspasen (Hamann et al., 2007) einen weiteren Ansatzpunkt für die Apoptoseforschung in P. anserina dar. Die molekularbiologische Analyse dieses Proteins wurde aufgrund dieser Proteomanalyse im Arbeitskreis aufgenommen (Dissertation D. Brust). Ein weiteres Protein, das in stark erhöhter Menge in den Proteinisolaten identifiziert wurde, ist PaMTH1. Im Rahmen der vorliegenden Arbeit wurden die Struktur und die Funktion dieser neu identifizierten differentiell-gebildeten Methyltransferase während der Alterung in P. anserina mit Hilfe molekularbiologischer, biochemischer und physiologischen Analysen untersucht. 3. Charakterisierung von PaMTH1 Im Rahmen von Northernblot-Analysen wurde gezeigt, dass die PaMth1-Transkriptmenge in drei unabhängigen alten Wildstämmen im Vergleich zu den entsprechenden jungen Wildtsämmen deutlich erhöht ist. In einer Westernblot-Analyse von Gesamtproteinen und Mitochondrien aus jungen und seneszenten Wildstämmen wird der seneszenzspezifische Anstieg der Proteinmenge verifiziert. Die genauere Einordnung von PaMTH1 in die Klasse I der Methyltransferasen und die Ergebnisse der Analyse der Substratspezifizität geben einen Hinweis auf eine Schutzfunktion durch die Verhinderung einer ROS-Entstehung unter der Beteiligung von Substanzen mit einer Catecholgruppe. Die Ergebnisse der Analyse der Modulation der PaMth1-Expression in P. anserina deuten ebenfalls auf eine Schutzwirkung von PaMTH1 hin: PaMth1-Überexpressionsstämme zeigen eine verbesserte Wuchsrate auf stress-induzierenden Medien, weniger carbonylierte Proteine und vor allem eine verlängerte Lebensspanne ohne physiologische Nachteile im Vergleich zum Wildstamm. Dagegen lebt die PaMth1-Deletionsmutante kürzer und wächst schlechter auf ROS-induzierenden Medien, sie zeigt allerdings keine erhöhte Menge von carbonylierten Proteinen im eindimensionalen „Oxyblot“. Die beobachtete Lebensspannenverkürzung der PaMth1-Deletionsmutante wird jedoch durch die Reversion dieser Stämme wieder aufgehoben, sodass die Hypothese des Schutzes vor der ROS-Generierung durch die Methylierung von Dihydroxylgruppen anhand der erhaltenen Daten unterstützt wird.
The attention on the protein PURA has increased recently following the discovery of the rare PURA Syndrome. This neurodevelopmental disorder is caused by de novo mutations in the PURA gene. Notably, our collaborators could show that the protein PURA can bind DNA and RNA in vitro. As a result, I was motivated to explore PURA's cellular RNAbinding activity. Furthermore, I inquired on the connection of PURA-RNA binding to the cellular effect of a reduction of functional PURA as present in PURA Syndrome patients.
To investigate the binding of PURA and the impact of PURA de ciency on cellular RNA and protein expression, I performed an integrative computational analysis of multimodal data from complementary high-throughput experiments. An essential component was the examination of UV Crosslinking and immunoprecipitation (CLIP) experiments, which can query the global RNA-binding behaviour of a given protein in a cellular context. As the processing and analysis of CLIP data are rather complex, I introduce an automated command line tool for the processing of CLIP data named racoon_clip as part of this dissertation. Therefore, this dissertation comprises two major segments. Firstly, I describe the implementation and usage of racoon clip for CLIP data analysis. Secondly, I discuss my research on the protein PURA, demonstrating its global RNA-binding properties, the effects of PURA depletion and its association with neuronal functions and P-bodies, among others.
racoon_clip is a command line application that I have developed for processing of individualnucleotide resolution CLIP (iCLIP) and enhanced CLIP (eCLIP) experiments - two of the most commonly used types of CLIP experiments - in a comparable and user-friendly way.
For this, I built racoon_clip as an automated work how that encompasses all CLIP processing steps from raw data to single-nucleotide resolution crosslink events. racoon_clip is available as a command line tool that users can run with a single command. The work how is implemented with Snakemake work how management providing computational advantage tages including parallelisation, scalability and portability of the work how. The main task of racoon_clip is to extract single-nucleotide crosslink events from iCLIP, iCLIP2, eCLIP and similar data types. To strike a balance between being highly customisable and easy to use, racoon_clip supplies pre-set options for the most common types of experiments.
Additionally, it is possible for users to create a custom setup of barcode and adapter architectures, which allows them to use the software for other types of CLIP data. While accounting for the different architectures in the reads, the performed central processing steps remain the same. This leads to a high degree of comparability between the different experiment types, which I demonstrate in the exemplary processing of U2AF2 iCLIP and eCLIP data. Taken together, I am confident that racoon_clip will be beneficial to numerous researchers interested in RNA-Protein interactions as it offers easily accessible processing for CLIP data and enhances the comparability of multiple CLIP datasets across di erent experiment types.
In the second part of this dissertation, I focus on the cellular function of the RNAbinding protein PURA. Through in-depth computational analysis of one iCLIP data set of endogenous PURA and two iCLIP data sets of overexpressed PURA in HeLa cells, I establish that PURA is a global RNA-binding protein. It preferentially binds RNAs in either the coding sequence (CDS) or the 3' untranslated region (3'UTR) of mature protein-coding transcripts by recognising a Purine-rich degenerated sequence motif. Even though overexpression of PURA results in less specific binding behaviour, the same overall binding patterns as from endogenous PURA persist. Overall characteristics of PURA binding remain similar in three distinct PURA iCLIP data sets with and without PURA overexpression.
To learn about the molecular consequences of a depletion of functional PURA in a cellular context, I used a 50% reduction of PURA in HeLa cells as a model for the heterozygous loss of PURA in PURA Syndrome and evaluated its impact on global RNA and protein expression. The results demonstrate that PURA depletion globally a ects RNA and protein expression. Additionally, I integrate PURA RNA binding with the changes in expression of RNAs and proteins in the context of PURA depletion. This reveals 234 targets of PURA that are bound by PURA and are impacted at both RNA and protein levels by the PURA protein. RNAs that are bound by PURA or change in abundance upon PURA depletion are enriched in neuronal development factors, RNA lifecycle regulators, and mitochondrial factors, among others. Consistent with a possible role of PURA in neuronal transport, there is considerable overlap between PURA bound transcripts and transcripts, that are transported to the dendritic end of neurons.
Notably, there is a link between PURA and P-bodies, as documented by the enrichment of PURA-bound RNAs in both the P-body and stress granule transcriptome. Further, PURA was found by our collaborators to be localised within P-bodies and P-body numbers were strongly reduced in cells that are depleted of PURA. This absence might be attributed to the downregulation of the proteins encoded by the PURA targets LSM14A and DDX6 as both of them were previously identified as essential for P-body formation.
Overall, the reduction of P-body numbers in PURA depletion, the neuronal function of PURA, and its association with mitochondria and RNA lifecycle regulation may indicate the cellular foundation of both PURA Syndrome and related neuronal diseases.
In summary, I present a versatile and user-friendly computational tool for the analysis of CLIP data. Subsequently, I conduct a thorough computational analysis of CLIP and other high-throughput data in the context of the RNA-binding protein PURA, which offers valuable insights into the cellular functions of PURA. These insights advance our understanding of the impact of PURA loss in PURA Syndrome and other disease contexts.
SIVsmmPBj-derived lentiviral vectors are capable of efficient primary human monocyte transduction, a capacity which is linked to the viral accessory protein Vpx. To enable novel gene therapy approaches targeting monocytes, in this thesis it was aimed to generate enhanced lentiviral vectors that meet the required standards for clinical applications with respect to gene transfer efficiency and safety. The vectors were tested for their suitability in a relevant therapeutic gene transfer approach. At first, it was investigated whether vectors derived from another Vpx-carrying lentivirus reveal the same capacity for monocyte transduction as SIVsmmPBj-derived vectors. A transduction experiment using HIV-2-derived vectors in comparison to PBj-derived vectors revealed a comparable transduction capacity, thus disproving the assumed uniqueness of the PBj vectors. The further generation and analysis of expression constructs for the vpx genes of HIV-2 and SIVmac demonstrated a similar functionality in monocyte transduction as the Vpx of PBj. As VpxPBj, both Vpx proteins facilitated monocyte transduction of a vpx-deficient PBj-derived vector system. For the generation of enhanced SIVsmmPBj and HIV-2 vector systems, only the transfer vectors were optimized, since the packaging vectors available already meet current standards. At first, several modifications were introduced into an available preliminary PBj-derived transfer vector by conventional cloning. The modifications included insertions of cPPT/CTS and WPRE as well as the deletions of the remaining pol sequence, the second exons of tat end rev, and the U3-region within the 3’LTR to generate a SIN vector. Thus, beside safety enhancement, the vector titers were also increased from 9.1x105 TU/ml achieved after concentration with the initial transfer vector up to 1.1x107 TU/ml with the final transfer vector. The PBj vector retained its capability of monocyte transduction when supplemented with Vpx. This conventional method of vector enhancement is time-consuming and may result in only sub-optimal vectors, since it depends on the presence of restriction sites which may not allow deletion of all needless sequences. Moreover, mutations may accumulate during the high number of cloning and amplification steps. Therefore, a new and easier method for lentiviral transfer vector generation was conceived. Three essential segments of the viral genome (5‘ LTR, RRE, ΔU3-3’ LTR) are amplified on the template of the lentiviral wild-type genome and fused by Fusion-PCR. Further necessary elements namely the cPPT/CTS-element, MCS, and PPT are included into the resulting vector by extension of the nucleotide primers used for the PCRs. The amplified and fused vector-scaffold can easily be integrated into a plasmid backbone, followed by insertion of the expression cassette of choice. By applying this approach, two novel lentiviral transfer vectors, based on the non-human SIVsmmPBj and the human HIV-2, were derived. Vector titers achieved for PBj and HIV-2 vectors supplemented with Vpx reached up to 4.0x108 TU/ml and 5.4x108 TU/ml, respectively. The capacity for monocyte transduction was maintained. Thus, safe and efficient, state of the art HIV-2- and PBj-derived vector systems are now available for future gene therapy strategies. Finally, the new vectors were used to set up an approach for gene correction of gp91phox-deficient monocytes for the treatment of X-linked chronic granulomatous disease (xCGD). The administration of autologous, gene-corrected monocytes to counteract systemic and acute infections could lead to a decreased infection load, dissolve granulomas and therefore improve the survival rate of hematopoietic stem cell transplantation (HSCT) which is the current treatment of choice for this disease. First, methods for analysis of gp91phox function were established. Next, they were employed to demonstrate the capacity of monocytes, obtained from healthy humans or mice, for phagocytosis, oxidative burst, and Staphylococcus aureus killing. The in vivo half-life of murine monocytes in the bloodstream and their distribution to specific tissues was determined. Lastly, HIV-1 vectors were used to transfer the gp91phox gene into monocytes from gp91phox-deficient mice. This resulted in the successful restoration of the oxidative burst ability in the cells. In summary, the general suitability of the new vectors for treatment of CGD by monocyte transduction was demonstrated. The results of the mouse experiments provide the foundation for future challenge experiments to evaluate the capability of gene-corrected monocytes to kill off microbes in vivo.
Der suprachiasmatische Nucleus (SCN) des Hypothalamus enthält die zentrale innere Uhr der Säugetiere. Diese innere Uhr besteht aus einem Verbund von untereinander gekoppelten Neuronen, die durch ein intrinsisches molekulares Uhrenwerk eine selbsterhaltende Oszillation mit einer Periode von circa einem Tag (circadian) aufrechterhalten. Diese Oszillation dient dem Organismus als innere Uhr und muss, da ihre Periode nicht exakt 24 Stunden beträgt, durch Zeitgeber mit der Umwelt synchronisiert werden. Der wichtigste circadiane Zeitgeber ist das Licht. Die Lichtsignale gelangen von der Retina über den retinohypothalamischen Trakt (RHT) direkt zum SCN. Von dort werden circadiane Informationen an zentrale und periphere Effektoren weitergeleitet, unter anderem an das Pinealorgan, welches zyklisch das Hormon Melatonin als Dunkelheitssignal ausschüttet. Melatonin kann wiederum auf die circadiane Uhr zurückkoppeln und in einem engen, sensitiven Zeitfenster die Phasen des SCN verschieben. Neben diesem Regelkreis gibt es direkte Verbindungen vom SCN zum lateralen Hypothalamus (LH), der eine zentrale Rolle bei der Regulation des Schlafes und der Energiehomöostase innehat. Ein spezieller Neuronentyp des LH schüttet das Neuropeptid Orexin aus, das große Bedeutung bei der Schlafregulation und der Appetitbildung besitzt. Diese orexinergen Neurone projizieren in weite Teile des Gehirns, unter anderem in die Region des SCN, was aufgrund der bekannten Wechselbeziehungen zwischen circadianer Aktivität, Schlaf und Appetit auf eine Rückkoppelung auf das circadiane System schließen lässt. In der vorliegenden Arbeit wurden mit Hilfe von Multielektroden-Ableitungen (MEAs) die neuronalen Signale einzelner Zellen des SCN, die zusammen ein komplexes Netz für die Steuerung der Effektormechanismen bilden, analysiert. Es standen dabei folgende Fragestellungen im Vordergrund: Bieten primäre Zellkulturen von SCN-Neuronen ein ausreichendes Modell für die Untersuchung der zellulären Kommunikation und der neuronalen Ausgangssignale der circadianen Uhr? Wirken Zeitgebersignale direkt auf die primären Oszillatoren oder sind Phasenverschiebungen eine Eigenschaft des gesamten Netzwerkes im SCN? Besteht ein Einfluss der orexinergen Neurone des lateralen Hypothalamus auf die Uhren-Neurone im SCN? Auf Multielektrodenplatten kultivierte SCN Neurone sind spontanaktiv und zeigen über Tage und Wochen ausgeprägte circadiane Rhythmen in ihrer Spikerate. In der Regel sind diese Aktivitäts-Rhythmen einzelner Neurone nicht synchronisiert, obwohl die Zellkulturen zahlreiche synaptische Verbindungen und korrelierte Aktivität aufweisen Um die Interaktion der verschiedenen Neuronen innerhalb des Uhrennetzwerkes aufzuklären, wurde eine Methode basierend auf Kreuzkorrelationen entwickelt. Mit dieser Methode konnte gezeigt werden, dass korrelierte Aktivität in SCN-Zellkulturen verbreitet ist, die stabilen Oszillatoren davon jedoch immer ausgenommen waren, obwohl eine durchgehende Vernetzung innerhalb der Kulturen bestand. Somit bilden Zellkulturen von SCN Neuronen ein sehr heterogenes Netzwerk mit stabilen Oszillatoren, schwachen Oszillatoren und nicht rhythmischen Neuronen, das viele einzelne Uhren elastisch miteinander koppelt und mit verarbeiteten Informationen über äußere und innere Zustände versorgt. Stabile Oszillatoren können direkt durch Zeitgeber-Stimuli, wie zum Beispiel Melatonin, beeinflusst und in ihren Aktivitäts-Phasen verschoben werden. Die Phasen-Antwortkurven sind im Vergleich zu in vivo Untersuchungen jedoch variabler, was für eine starke Beteiligung des neuronalen Netzwerkes bei der Verarbeitung und Stabilisierung der Antworten auf die Zeitgeber-Stimuli spricht. Phasenverschiebungen als Reaktion auf einen Zeitgeber-Stimulus sind demnach eine Eigenschaft der einzelnen Oszillatorzelle, die aber durch die Interaktion der verschiedenen Uhren-Neurone in ein stabiles Ausgangssignal umgesetzt werden müssen. Kultivierte SCN-Neurone reagieren auf Applikation von Orexin A mit kurzzeitigen Änderungen der Spikerate, sowie mit deutlichen Phasenverschiebungen, die in Zellkulturen sehr variabel ausfallen, während sie in Ableitungen von organotypischen Hirnschnitten stabil und reproduzierbar sind. Dies unterstreicht wiederum die Bedeutung des neuronalen Netzwerkes im SCN. Orexin A scheint zwar direkt auf stabile Oszillatorzellen einzuwirken, aber auch auf die synaptische Übertragung zwischen den SCN-Neuronen. Sein Wirkungsmechanismus könnte dabei in einer Hemmung der GABAergen und in einer Verstärkung der glutamatergen synaptischen Übertragung liegen. Die Wirkung von Orexin A im SCN spricht für eine vielfältige Rückkoppelung des orexinergen Systems auf den circadianen Schrittmacher.
Die Zellwand von Arabidopsis thaliana enthält große Menge an Hemicellulosen und Pektinen, deren Bestandteile sich hauptsächlich von UDP-Glucuronsäure ableiten. Die Bildung von UDP-Glucuronsäure wird in Pflanzen überwiegend durch die UDP-Glucose Dehydrogenase (UGD) katalysiert, die UDP-Glucose unter der Bildung von NADH in UDP-Glucuronsäure umwandelt. Arabidopsis thaliana besitzt vier UGD-Gene und ein Pseudogen, welche starke Homologien zu Genen anderer bekannter pflanzlicher UDP-Glucose Dehydrogenasen zeigen. Mit Hilfe von Promotor::GUS-Reportergenpflanzen und real time-PCR-Analysen konnte in dieser Arbeit gezeigt werden, dass die vier UGD-Gene nicht nur in verschiedenen Geweben und zu unterschiedlichen Zeitpunkten der Morphogenese exprimiert werden, sondern auch in unterschiedlicher Stärke. Dabei scheint jedoch zu jedem Zeitpunkt der Morphogenese, bis auf die Samenentwicklung, eine der vier UGD-Isoformen in Arabidopsis thaliana exprimiert zu werden. Eine biochemische Charakterisierung der verschiedenen Isoformen zeigte einen sehr ähnlichen Km-Wert von ca. 43 µM für NAD+, während sich die Km-Werte für UDP-Glucose deutlich voneinander unterschieden (123 - 335µM). Alle Isoformen unterlagen einer feedback-Hemmung durch UDP-Xylose. Dabei war eine starke Hemmung durch UDP-Xylose korreliert mit einer hohen Affinität zu UDP-Glucose. Neben NAD+ konnten alle untersuchten Isoformen in geringem Maße auch NADP+ als Cofaktor verwenden. Allerdings verringerte sich die Enzymaktivität dadurch um etwa 80%. Alternative Zuckersubstrate konnten dagegen nicht umgesetzt werden. Die biochemischen Unterschiede zwischen den UGD-Isoformen und die differentielle Expression ihrer entsprechenden Genekönnten eine wichtige Rolle bei der Regulation der Zellwandbiosynthese spielen. Denn die irreversible Oxidation von UDP-Glucose zu UDP-Glucuronsäure durch UGD fungiert als eine der Schaltstellen, an welcher der Kohlenstofffluss derpflanzlichen Zelle in Richtung Zellwandbiosynthese gesteuert werden kann. Die im Rahmen dieser Arbeit durchgeführten Analysen von Einfach-oder Doppel-knock out-Mutanten, bei denen durch eine T-DNA-Insertion ein oder zwei UGD-Gene ausgeschaltet waren, zeigte dementsprechend auch eine veränderte Zellwandzusammensetzung bei den Mutanten deltaUGD2, deltaUGD3 und deltaUGD1x deltaUGD4 und eine veränderte Funktion der Stomata bei deltaUGD1x deltaUGD4. Insgesamt waren die Phänotypänderungen gegenüber dem Wildtyp bei der Doppelmutante deltaUGD1x deltaUGD4 wesentlich ausgeprägter als bei den Einfachmutanten, was daran liegen könnte, dass der Ausfall eines UGD-Gens durch ein anderes kompensiert wird. Dafür sprechen auch die Ergebnisse der real time-PCR-Analyse, in der die Expression von UGD1, 2, 3 und 4 in sechs Tage alten Keimlingen des Wildtyps und der knock out-Mutanten untersucht wurde. Dort konnte nachgewiesen werden, dass sich das Ausschalten eines oder mehrerer UGD-Gene auf die Expression der übrigen UGD-Gene auswirkt.
The mammary gland of mice serves as a model system for studying differentiation in an adult animal. With the beginning of pregnancy the mammary epithelial cells undergo functional differentiation to produce milk for nourishment of the young. The transcription factor STAT5 mediates the cytokine-induced induction of the milk proteins during pregnancy and lactation in response to the lactogenic hormone prolactin. In addition to transcription factors that mediate transcription of their target genes by recruitment of the general transcription machinery to the DNA-regulator regions, specific post-translational modifications on the N-terminal tails of histones also influence expression. These histone modifications can affect chromatin structure, which is a main control barrier to transcription, by directly altering accessibility of the chromatin and by providing binding surfaces for protein complexes that can further modulate chromatin structure and regulate transcription. In this work N-terminal histone modification marks that associate with open, permissive and repressed chromatin where investigated in different regions of two milk protein genes during mammary gland development. Using the chromatin-immunoprecipitation (ChIP) assays increased acetylation of histone H3 and H4 at the 5’ region, promoter and transcribed regions of β-casein and whey acidic protein (WAP) gene were observed during pregnancy and lactation when these genes are expressed. The presence of these histone marks, which are associated with a relaxed chromatin structure, correlates with the recruitment of STAT5A and STAT5B to the promoter containing regulatory regions as well as the detection of the phosphorylated RNA polymerase II in the transcribed gene region. Both di- and tri-methylation of histone H3 lysine 4, that mark permissive and active chromatin respectively, were enriched in tissue from pregnant and lactating mice. In comparison tri-methylation of histone H3 lysine 27, a mark associated with repressed chromatin, could be observed during all stages of mammary gland tissue investigated, but appears slightly elevated in the tissue from virgin mice when β-casein and WAP are not expressed. Together these results illustrate that the expression of the two milk proteins genes at distinct stages of mammary gland differentiation correlate with specific changes in histone modifications. In mammary gland tissue STAT5A is important for the mammary gland epithelial cell differentiation and survival during lactation. Yet many genomic target regions that STAT5A actually bind and which are involved in regulation of gene expression during lactation still remain unknown. Therefore, the second part of this thesis was focused on the identification of novel STAT5-binding sites that are differentiation specifically bound by STAT5A in mammary gland tissue during lactation. In summary, the results demonstrate that the ChIP cloning method was employed successfully for the cloning of a STAT5A library and the identification of new STAT5 targets in mammary gland tissue from lactating mice. Nine of the newly identified STAT5-binding targets were verified to differentiation specifically bind STAT5A and STAT5B in vivo during pregnancy and lactation. Even though the selection of the tested clones was biased towards STAT5-binding sites near or at known genes and for multiple STAT5 binding sites, only one out of the nine validated STAT5-binding regions is located in a traditional defined proximal promoter. Except for two STAT5-binding regions, which are located at least 10 kb from the next annotated known gene, six are located in the intronic regions of annotated mRNA or EST transcripts. Three, out of four verified STAT5-binding regions tested in reporter gene assays for functionality, display the ability to drive reporter gene activity in a STAT5 dependent manner. This transcriptional activity is due to the STAT5-binding sites within the cloned regions as determined by mutational analysis. Of special interest is a STAT5-binding region that contains one STAT5 and three STAT-like sites within a 339 bp region that is evolutionary conserved by approximately 80% between the mouse and human genome. This STAT5-binding region lies about 62 kb 5 prime of the nuclear factor I/B gene. The expression of the NFI/B mRNA transcript correlates with the in vivo association of STAT5A to the conserved region during the mammary gland differentiation. Together, these results suggest that this STAT5-binding might be a cis-regulatory region that potentially mediates STAT5 induced NFI/B gene expression in mice during lactation.
In order to form an organ, cells need to take up specialized functions and tasks. Cellular specialization is guided by an interplay of chemical signals and physical forces, where one influences the other. One aspect in cellular identity is its shape, which e.g. defines how susceptible the cell may be to intercellular signaling or in which section of the cell cycle it is and therefore can tell us about its current state. Shape changes are introduced by motor proteins that are controlled and activated in a locally confined manner. For my thesis, I was interested to understand better how cellular shape and geometry impacts downstream cell and organ development. What happens if a cell cant transition to a specific shape? How does it affect tissue structure? How does it affect further development?
One regulator of motor proteins like non-muscle myosin is Shroom3, which recently has been been shown to be expressed and involved in the development of the zebrafish lateral line organ (1 ). Development of the lateral line occurs through a migrating cluster of initially about 150 cells, the posterior lateral line primordium (pLLP), which migrates from the anterior (head) to the posterior (tail) while depositing cell clusters in a regular pattern. Literature on development of the lateral line suggests that in order for a cell cluster to be deposited from the pLLP, rosette formation is a key requirement. Therefore our expectation from the shroom3 mutant was that the number of clusters deposited was significantly reduced. To our surprise, when we first inspected the end of migration lateral line phenotype we found many individuals with a significant increase in cell clusters deposited.
This made us re-think the role of Shroom3 during rosette assembly and the processes its involved in.
To study the effects of Shroom3 on lateral line development, a mutant line was generated and crossed with various transgenic lines which express fluorescently labeled proteins that locate to organelles such as the plasmamembrane or the nucleus. Following, the mutant with its fluorescent labels was microscopically imaged under different conditions to quantify and analyze various cell-morphometric features. Even though the zebrafish is a popular model organism and its perfectly suited for developmental biology and advanced microscopy, there were no methods that would allow for a standardized and more automated pipeline of data acquisition and processing.
Therefore, in order to accurately quantify the morphogenic processes Shroom3 is involved in, I developed a new toolset that significantly improved and facilitated my research. The toolset consists of (1) a new sample mounting method that is based on a 3D agarose gel that increases the number of embryos that can be mounted and imaged at once and speeds up the imaging process significantly (2) for subseqent image analysis I developed four programs that automate the process and therefore make the results much more reproducible and the analysis much more efficient. The first program is used for end of migration analyses, to deduce the pattern, count and size of Lateral Line cell clusters. The second is used not for end of migration, but for migration analyses (on timelapse recordings). Besides this it also prepares the images for more advanced downstream migration analyses and allows to analyse fluorescence signal on a second channel. The third program is used to analyse the pLLP only at high spatial resolution and to deduce the cell count, 3D cell morphometrics (like the volume) and cell orientation. The fourth program finally is used downstream of the second and third program and is capable of detecting and comparing them with the look of wildtype rosettes.
Here I show that in absence of Shroom3 rosette formation in the migrating pLLP is destabilized leading to facilitated cell cluster deposition and I show how this might be related to traction forces due to a possible interdependence of pLLP acceleration and speed of migration. Furthermore I show that apical constriction and rosette formation is not blocked in Shroom3 deficient embryos, but that larger rosettes are fragmented into many smaller ones. Finally, I give an outlook on how the absense of Shroom3 and hence the absense of morphological changes may deregulate gene transcription by elevating the levels Atoh1a, a transcription factor necessary for hair cell development.
My results and methodology demonstrate the importance of morphology in guiding developmental processes and how rather small morphological changes on the cellular level can impact further development significantly. My work also shows how powerful modern genetics, imaging and image analysis are and how diverse they are in terms of range of questions they are capable of answering. The methods and tools I developed prepare the ground for at least three quarters of the analyses I carried out and together with the documentation and data I provide, they are highly reproducible. In that regard I am especially happy that one of my developments, an improved sample preparation method, is already used by many different labs all over the world helping them to make their results more reproducible.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
Die Onkogenese geht mit einer Deregulation des Zellzyklus einher. Dabei spielt unter anderem die Regulation verschiedener krebsrelevanter Transkriptionsfaktoren eine wichtige Rolle. Ein Interaktionspartner und Regulator vieler dieser Faktoren ist das LIM-only Protein FHL2. Es ist bereits bekannt, dass sich der FHL2-Status zwischen normalen und entarteten Zellen in allen bisher untersuchten Geweben unterscheidet. Im Rahmen dieser Arbeit konnte erstmals gezeigt werden, dass dies auch im Brustgewebe der Fall ist. FHL2 wird in fast allen aggressiven Mammakarzinomen überexprimiert, nicht aber im Normalgewebe und nur schwach in nicht-invasiven "DCIS". Dies weist darauf hin, dass die FHL2-Menge mit der Aggressivität des Tumors korreliert. Weiterhin konnte hier zum ersten Mal nachgewiesen werden, dass FHL2 in die Zellzyklusregulation involviert ist. Am G1/S-Übergang kann FHL2 die Cyclin D1-Expression induzieren, was letztendlich zu einer Phosphorylierung des RB-Proteins und zum Eintreten der Zelle in die S-Phase führt. Wichtiger aber ist die hier gezeigte FHL2-abhängige Induktion von p21CIP/WAF, ein Zellzyklusinhibitor, der unter anderem auch in der G2/M-Kontrollpunkregulation involviert ist und normalerweise über p53 reguliert wird. Diese Induktion resultiert dort in einem verlangsamten Kontrollpunktübergang, wogegen eine Reduktion des FHL2-Gehalts einen beschleunigten G2/M-Übergang zur Folge hat. Zusätzlich zeigten Expressionsanalysen mit synchronisierten Brustkrebszellextrakten dass FHL2 zellzyklusabhängig exprimiert wird, mit einem Maximum am G2/M-Kontrollpunkt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die p21-Expression in den hier verwendeten Brustkrebszelllinien p53-unabhängig ist und ausschließlich von FHL2 abhängt. Hierbei wird die FHL2-abhängige p21 Expression wahrscheinlich über die Aktivierung des c-jun-Transkriptionsfaktors im MAPK-Signaltransduktionsweg induziert. In vivo und in vitro Interaktionsstudien haben eine Interaktion von FHL2 mit c-jun gezeigt, wobei die Interaktion über die ersten beiden LIM-Domänen vermittelt wird. Der FHL2-c-jun-Komplex bindet an die AP-1-Sequenz innerhalb des p21-Promotors und induziert dadurch p21. Dies führt zu einer Inhibition verschiedener CDE/CHR-regulierter Proteine wie CDC25C oder Plk1 und zu einer verzögerten Zellzyklusprogression. In diesem Zusammenhang konnte im Rahmen dieser Arbeit gezeigt werden, dass die FHL2-Expression nicht nur zu einer verlangsamten Proliferation führt, sondern auch zur Fähigkeit der Zelle zum zellmatrixunabhängigen Wachstum beisteuert. Es scheint auf den ersten Blick widersprüchlich, dass FHL2 für einen intakten G2/M-Kontrollpunkt und eine geringere Proliferationsrate sorgt, gleichzeitig aber zur Tumorentwicklung beiträgt. Es ist allerdings bekannt, dass Tumore ihr Wachstum verlangsamen bevor sie metastasieren. Auch führt ein erhöhter p21-Gehalt im Cytosol zu einer Inhibition der Apoptose, einer weiteren Eigenschaft von Tumoren. FHL2 ist daher ein signifikanter Faktor in der Onkogenese des Mammakarzinoms und aufgrund der differentiellen Expression in vielen Tumoren ein interessantes Ziel für Krebstherapien.
Integral membrane proteins (IMPs) account for 20-40% of all open reading frames in fully sequenced genomes and they are target of approximately 60% of all modern drugs. So far, cellular expression systems are often very insufficient for the high-level production of IMPs. Toxic effects, instability or formation of inclusion bodies are frequently observed effects that prevent the synthesis of sufficient amounts of functional protein. I have successfully established an individual cell-free (CF) expression system to overcome these IMP synthesis difficulties. The CF system was established in two different expression modes. If no hydrophobic compartment is provided, the IMPs precipitate in the reaction mixture. Interestingly, these insoluble proteins are found to differ from inclusion bodies as they readily solubilize in mild detergents and the bacterial small multi drug transporter EmrE, expressed in the insoluble mode was shown to reconstitute into liposomes in an active form. Alternatively, IMPs can be synthesized in a soluble way by supplementing the CF system with detergents. A comprehensive overview of 24 commonly used detergents was provided by analyzing their impact on the CF system as well as their ability to keep three structurally very different proteins in solution. The class of long chain polyoxyethylene-alkyl-ethers turned out to be most suitable for soluble expression of a-helical EmrE, the bacterial b-barrel type nucleoside transporter Tsx and the porcine vasopressin receptor type 2, resulting in several mg of protein per mL of reaction mixture. So far IMPs have almost completely been excluded from solution nuclear magnetic resonance (NMR) analyses. I could demonstrate that CF expression enables efficient isotopic labeling of IMPs for NMR analysis and further facilitates selective labeling strategies with combinations of 13C and 15N enriched amino acids that have not been feasible before. Four different G-protein coupled receptors (GPCRs) were successfully CF expressed in preparative scale and for the human endothelin B receptor (ETB), ligand binding ability was observed. A series of truncated ETB derivatives containing nested terminal deletions have been CF produced and functionally characterized. The core area essential for Endothelin-1 binding as well as a central region responsible for ETB oligomer formation was confined to a 39 amino acid fragment including the proposed transmembrane segment 1. The binding constant (KD) of ETB was determined to 6 nM for circular ET-1 by SPR and 29 nM for linear ET-1 by TIRFS. This data indicate a large potential of the established individual CF expression system for functional IMP synthesis.