Characterisation of the tissue-specific localization of the molecular chaperon Mdg1 and identification of cell-compartement-specific proteindomains

Deckblatt-Impressum persönlicher Dank Inhaltsverzeichnis Abkürzungsverzeichnis Einleitung Fragestellung und Zielsetzung Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Literaturverzeichnis Anhang Danksagung SelbständigkeitserklärungMolekulare Chaperone, auch als Hitzeschockproteine bezeichnet, kommen in allen Zellkompartimenten vor und sind an Proteinfaltung, Degradation und Proteintransport beteiligt. Eine Veränderung der subzellulären Lokalisation bei wechselnden äußeren Bedingungen kann bei einigen Chaperonen beobachtet werden. Auch in der Entwicklung der Organismen nehmen sie eine zentrale Rolle ein. Ziel dieser Arbeit war es, die subzelluläre Lokalisation in COS7-Zellen des Mdg1-Proteins, einem molekularen Chaperon, und die dafür verantwortlichen Proteindomänen zu charakterisieren. Außerdem sollte die Lokalisation des Mdg1-Proteins in verschiedenen Organen der adulten Maus sowie in verschiedenen Entwicklungsstadien charakterisiert werden. Dies wurde mit molekular-, zellbiologischen, proteinbiochemischen und immunhistochemischen Methoden realisiert. Dabei konnte der Domäne CSGQ, welche sich am C-terminalen Ende von Mdg1 befindet, eine entscheidende Rolle bei der Lokalisation an Membranen unter Kontrollbedingungen zugeordnet werden. Für die Assoziation am Zytoskelett sind die Aminosäurebereiche 96 bis 125 und 180 bis 222 des C-Terminus verantwortlich. Durch Hitzeschock transloziert Mdg1 in den Zellkern und reichert sich in den Nukleoli an, wofür ebenfalls der C-terminale Sequenzabschnitt CSGQ verantwortlich ist. Bei der Translokation sind außerdem die vorderen und hinteren etwa 30 Aminosäuren (AS 96 bis 125 und AS 180 bis 222) von entscheidender Bedeutung. Bei der Untersuchung der Lokalisation von Mdg1 in verschiedenen embryonalen und fetalen Entwicklungsstadien sowie adulten Mäusen konnte beobachtet werden, dass Mdg1 während der gesamten Entwicklung vor allem in sich differenzierenden Zellen vorkommt. Im adulten Organismus ist das Mdg1-Protein besonders in sekretorisch aktiven Zellen nachweisbar. Die Ergebnisse lassen Rückschlüsse auf eine mögliche Funktion von Mdg1 zu. Mdg1 kommt vor allem in sich differenzierenden und sekretorisch aktiven Zellen vor, welche sich in der G0-Phase befinden, in der keine Zellteilungen stattfinden. Somit ist eine Beteiligung von Mdg1 am Zellzyklus denkbar. Mdg1 könnte einen Zellzyklusarrest bewirken und dadurch Differenzierung und Sekretion ermöglichen.Molecular Chaperones, also known as heat shock proteins, are localized in all cell compartments and are important for protein folding, degradation and protein transport. Some chaperones change their subcellular localization due to alterations in environment. They are also very important for the development of organisms. The aim of this thesis was the characterization of the subcellular localization of Mdg1, a molecular chaperon, and the responsible protein domains. In addition to that the localization of Mdg1 was characterized in adult mice and different stages of development. The experiments were realised by using molecular and cell biological, proteinbiochemical and immunohistochemical methods. The studies showed that the c-terminal domain CSGQ has an important role for the localization on membranes. The amino acids 96 to 125 and 180 to 222 are responsible for the association with the cytoskeleton. Under heat shock Mdg1 translocates into the nucleus and accumulates in the nucleoli. The c-terminal motif CSGQ is also responsible for this translocation. Furthermore the first and the last 30 amino acids (96 to 125 and 180 to 222) are important for the translocalization into the nucleoli. The investigation of the localization of Mdg1 in different stages of development and in adult mice showed that this protein could be recognised in all stages especially in differentiating cells. In adult mice Mdg1 could be detected particularly in secretory cells. The results lead to conclusions about the function of Mdg1. As the protein is mainly found in differentiating and secretory cells it could participate in the cell cycle, because these cells are in the rest phase. So Mdg1 could cause a cell cycle arrest to make a differentiation and secretion possible

Changes of inhibitory micronetworks in the epileptic hippocampus and their response to anticonvulsant drugs

With around 50 million people worldwide suffering from chronic epilepsy, it is one of the most common neurological disorders. However, despite extensive research in this area, the actual network mechanisms involved in the generation of seizures are still not fully understood. This lack of insight is especially important given that 30 % of patients still cannot be treated sufficiently with the available antiepileptic drugs. Outstanding alterations in the epileptic brain are the loss of inhibitory interneuron subtypes and a reduction in GABAergic responses. They suggest that the pathological hyperexcitability characteristic of epilepsy is related to a reduction in GABAergic inhibition. However, the role of GABAergic inhibition is far from limited to reducing neuronal excitability: inhibitory interneurons control spike timing of pyramidal cells, synchronize large populations of neurons and play a role in network oscillations that occur during different states of the brain. These different tasks of interneurons suggest that changes in the spatial and temporal profile of surviving interneurons will also strongly influence network. In the first part of my PhD thesis I therefore investigated whether the spatiotemporal profile of feed-back inhibition is changed in chronically epileptic rats using the pilocarpine model of epilepsy. I focused on the CA1 area of the hippocampus because this brain region is strongly affected in temporal lobe epilepsy, a subform of epilepsy with a high incidence of pharmacoresistance. Using electrical activation of inhibitory feed-back microcircuits in acute hippocampal slices, I could show that the spatiotemporal profile of inhibition provided by the surviving interneurons is altered in experimental epilepsy. As a result, the usually strong initial feed-back inhibition onto CA1 pyramidal cells was reduced. These data suggest marked changes in the dynamics of feed-back inhibition in chronic epilepsy that may be relevant for the initiation of seizure activity in the CA1 ensemble. In the second part of my thesis I investigated the effects of commonly used sodium channel blocking anticonvulsants on inhibitory microcircuits. GABAergic interneurons are able to fire at very high rates and should consequently be affected by these drugs. This however would cause a further reduction of GABAergic inhibition and thus increase excitability. My experiments revealed that, based on intrinsic and circuit properties, under these experimental conditions, both feed-forward and feed-back inhibition are not affected by these drugs, enabling them to fulfill their function. Furthermore, this study shows that the net effect of CNS drugs on the complex neuronal circuitry within the brain cannot be predicted by their action on individual cell types but also critically depends on the interplay of neuronal subtypes within this network

Spontaneous combustion of hydrogen

It is shown by the author's experiments that hydrogen which escapes to the atmosphere through openings in the system may burn spontaneously if it contains dust. Purely thermal reasoning can not account for the combustion. It seems to be rather an electrical ignition. In order to determine whether the cause of the spontaneous ignition was thermo-chemical, thermo-mechanical, or thermo-electrical, the experiments in this paper were performed

Chromatin Control by the Human Cytomegalovirus Immediate-Early 1 Protein

Human cytomegalovirus (hCMV), one of eight human herpesviruses, establishes lifelong "latent" infections in 40-100% of people worldwide. HCMV replication following primary infection or reactivation is known for causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals, but preventive and therapeutic options are still limited. One potential candidate for the development of new antiviral drugs or a vaccine is the immediate-early (IE) 1 protein of hCMV. This protein is a crucial regulator of viral and cellular gene expression and has been shown to interact with chromatin. For chromatin binding, the IE1 protein exhibits two adjacent core histone interacting regions with distinct binding specificities. One of them is the so-called "chromatin tethering domain" (CTD), a 16 amino-acid sequence (amino acids 476-491) at the IE1 carboxy-terminus, which was recently shown to bind to the acidic patch formed by histone H2A and H2B on the nucleosomal surface to which several other viral and cellular proteins bind as well. The latency-associated nuclear antigen 1 (LANA) encoded by the Kaposi’s sarcoma-associated herpesvirus (KSHV) binds to the acidic patch in a way similar to the IE1-CTD and was shown to regulate chromatin compaction, viral genome maintenance and the cellular DNA damage response (DDR) via this interaction. Based on the similarities to LANA and the fact that nucleosome targeting by IE1 is dispensable for productive replication of hCMV, we hypothesized that the two viral proteins may serve analogous functions during latency of their respective viruses. In this thesis, I focused on defining chromatin binding sites of IE1 and uncovering the function of nucleosome targeting by IE1 with respect to genome maintenance and DDR. To identify chromatin binding sites of IE1 on both the viral and cellular genome, I generated primary human fibroblasts (MRC-5 cells) permissive to hCMV in which expression of HA-tagged IE1, untagged IE1, and HA-tagged CTD-deleted IE1 (IE1₁₋₄₇₅) can be synchronously induced. Chromatin immunoprecipitation coupled to next generation sequencing (ChIP-seq) experiments using these cells revealed that IE1 broadly binds to the host genome in a CTD-dependent manner. The protein appears to be enriched at transcription end sites (TES) and excluded from the promoter regions of human genes at the transcription start sites (TSS), which may be due to differences in the nucleosomal load at these sites. Broad binding of IE1 was also observed across the viral genome, but here four binding peaks were identified. During viral latency IE1 may use nucleosome binding as a mechanism to tether the viral genome to host chromosomes similar to what has been observed for LANA. Against all controversies regarding the presence of IE1 during non-productive stages of infection, I could identify full-length IE1mRNA and protein and IE2 protein in a latently hCMV-infected monocytic cell line (THP-1 cells). Time-course analysis of viral genome levels in these cells showed that chromatin binding by IE1 is necessary for cyclic viral DNA replication events during latency through which the virus probably ensures viral genome maintenance. Other results demonstrate that IE1 reduces the nucleosomal load on the viral and host genome in a CTD-independent manner, perhaps to reduce chromatin compaction and promote transcription. Similar to LANA, chromatin binding by IE1 also affects the DDR outcome. IE1 blocks H2A(X)K13/15 and H2A(X)K118/119 ubiquitination by binding to the nucleosomal acidic patch and thereby seems to diminish DNA double-strand break repair by non-homologous end joining. These results indicate that IE1 broadly interacts with viral and cellular chromatin via the nucleosome surface and suggest that the IE1-nucleosome interaction serves an important role in controlling viral genome maintenance and the outcome of the DDR in hCMV-infected cells

Messenger RNA Expression of Selected Factors at Different Sites of the Bovine Endometrium Associated With Uterine Health

Recent studies have elucidated the role of several pro-inflammatory factors as mediators of inflammatory processes in the bovine endometrium. Only few studies, however, have analyzed samples collected from different regions of the uterus of the same animal. In this study, we tested the hypothesis that on a molecular level, clinical endometritis is characterized by inflammatory responses spread over the entire endometrium. Furthermore, we assume that subclinical endometritis is described by an inflammation of local regions of the uterus. Therefore, the objective of this study was to assess the mRNA expression of uterus-associated pro-inflammatory factors at five pre-defined endometrial sites, i.e., corpus uteri, left horn base, left horn tip, right horn base, and right horn tip, in cows with clinical and subclinical endometritis and in healthy controls. We analyzed the mRNA expression of interleukin 1 alpha, interleukin 1 beta, C-X-C motif chemokine ligand 8, prostaglandin-endoperoxide synthase 2, protein tyrosine phosphatase receptor type C, carcinoembryonic antigen related cell adhesion molecule 1, and mucin 4 and 16. Based on vaginoscopy and endometrial cytology (>= 5% polymorphonuclear neutrophils) between 28 to 34 days in milk, 18 Simmental cows were categorized in clinical endometritis group (n = 7), subclinical endometritis group (n = 4), and healthy group (n = 7). In general, the analyses revealed a great variation of mRNA expression between sites and animals. Differences were found between different uterine health statuses, but the variation between the sampling sites within the groups was not significant (P > 0.05). This indicates that inflammatory processes at the end of the postpartum period can be regarded as multi-focal or spread throughout the uterus independent from the uterine health status

Cytological endometritis diagnosed at artificial insemination in repeat breeder dairy cows

This study aimed to (i) assess the prevalence of cytological endometritis (CYTO) diagnosed at artificial insemination (AI); (ii) evaluate the effect of CYTO on the pregnancy outcome of the same AI sample; and (iii) determine the risk factors associated with CYTO diagnosed at AI in repeat breeder (RB) dairy cows. We analysed the productive and reproductive performances of 146 RB Holstein-Friesian cows. To obtain a CYTO sample at AI, we used the cytotape technique. Generalized mixed effect models were computed to find the risk factors associated with the pregnancy and CYTO outcome. Based on 1% PMN cut-off point, the CYTO prevalence at AI in RB cows was 25.3%. The overall pregnancy at AI was 44.2%. The conception rate in CYTO-positive (n=37) RB cows was 29.7% versus 49.5% for CYTO-negative (n=109) cows. A RB cow diagnosed CYTO positive at AI had 0.47 [odds ratio (OR)] odds to become pregnant in comparison with a CYTO-negative cow. Cows that produced more milk than their counterparts in this study had increased odds (OR=1.01) to be CYTO positive at AI. A novel risk factor positively associated with CYTO diagnosed at AI in RB cows was the level of daily milk urea (OR=1.11). To conclude, CYTO at the moment of AI had a significantly negative effect on the pregnancy outcome in RB dairy cows. However, as only one of fourth of RB cows is affected with CYTO at AI, it may not be considered a key element associated with the RB syndrome

Prekäre Lebenslagen von Kindern und Jugendlichen - Herausforderungen für die Kinder- und Jugendhilfe. Expertise zum 9. Kinder- und Jugendbericht des Landes Nordrhein-Westfalen

[Diese Expertise] ist als Beitrag zu einer landesspezifischen Sozialberichterstattung über Kinder, Jugendliche und junge Erwachsene sowie über das Leistungsspektrum der Kinder- und Jugendhilfe zu verstehen. Damit ist sie nach Anspruch und Methode zwischen Wissenschaft, Forschung und Politik zu verorten. Sie basiert großteils auf einer empirisch-quantitativen Datengrundlage [und rückt] auf einer deskriptiven Basis relevante Aspekte der Bedingungen des Aufwachsens, der gegenwärtigen Lebensverhältnisse und der Teilhabechancen von Kindern und Jugendlichen in Nordrhein-Westfalen in den Mittelpunkt. Mit der Fokussierung auf prekäre Lebenslagen werden überwiegend die Lebensbedingungen und Teilhabechancen von Heranwachsenden aus schwierigen sozialen Verhältnissen betrachtet. … In der Expertise werden überwiegend diejenigen Ausschnitte der Lebensbedingungen der nachwachsenden Generation beleuchtet, die sich auf das Aufwachsen in öffentlicher Verantwortung beziehen. Infolgedessen geht es sowohl um eine Ausrichtung an den Akteuren selbst, d.h. den Kindern und Jugendlichen, als auch um eine Berücksichtigung der Institutionen, insbesondere der Einrichtungen der Kinder- und Jugendhilfe. (DIPF/Orig.

Impaired D-Serine-Mediated cotransmission mediates cognitive dysfunction in epilepsy

The modulation of synaptic plasticity by NMDA receptor (NMDAR)-mediated processes is essential for many forms of learning and memory. Activation of NMDARs by glutamate requires the binding of a coagonist to a regulatory site of the receptor. In many forebrain regions, this coagonist is D-serine. Here, we show that experimental epilepsy in rats is associated with a reduction in the CNS levels of D-serine, which leads to a desaturation of the coagonist binding site of synaptic and extrasynaptic NMDARs. In addition, the subunit composition of synaptic NMDARs changes in chronic epilepsy. The desaturation of NMDARs causes a deficit in hippocampal long-term potentiation, which can be rescued with exogenously supplied D-serine. Importantly, exogenous D-serine improves spatial learning in epileptic animals. These results strongly suggest that D-serine deficiency is important in the amnestic symptoms of temporal lobe epilepsy. Our results point to a possible clinical utility of D-serine to alleviate these disease manifestations