Diss. phil.-nat. Bern, 1983.

Diss. med. Bern, 1977. / SA aus: Biochimica et biophysica acta. - 489(1977), S. 58-63.

Universiẗat, Diss., 2004--Kiel. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Regensburg, Univ., Diss., 2007

Techn. Universiẗat, Diss., 2000--Berlin.

Bacterial functional membrane microdomains (FMMs) are membrane platforms that resemble lipid rafts of eukaryotic cells in certain functional and structural aspects. Lipid rafts are nanometer-sized, dynamic clusters of proteins and lipids in eukaryotic cell membranes that serve as signaling hubs and assembling platforms. Yet, studying these structures can often be hampered by the complexity of a eukaryotic cell. Thus, the analogous structures of prokaryotes are an attractive model to study molecular traits of this type of membrane organization. Similar to eukaryotic lipid rafts, the bacterial FMMs are comprised of polyisoprenoid lipids, scaffold proteins and a distinct set of membrane proteins, involved in signaling or secretion. Investigating bacterial FMMs not only contributes to the understanding of the physiological importance of FMMs in bacteria, but also helps to elucidate general principles of rafts beyond prokaryotes. In this work, a bacterial model organism was used to investigate effects of synthetic overproduction of the raft scaffolding proteins on bacterial physiology. This overexpression causes an unusual stabilization of the FMM-harbored protease FtsH and therefore the proteolytic targets of FtsH are not correctly regulated. Developmental defects and aberrances in shape are the consequence, which in turn negatively affects cell physiology. These findings may be adapted to better understand lipid raft processes in humans, where flotillin upregulation is detected along with development of neurological diseases. Moreover, it was aimed at understanding the FMM-proteome of the human pathogen Staphylococcus aureus. An in-depth quantitative mass-spectrometry analysis reveals adaption of the protein cargo during different conditions, while maintaining a distinct set of core FMM proteins. As a case study, the assembly of the type VII secretion system was shown to be dependent on FMM integrity and more specifically on the activity of the FMM-scaffold flotillin. This secretion system is important for the virulence of this pathogen and its secretion efficiency can be targeted by small molecules that inhibit flotillin activity. This opens new venues for non-conventional antimicrobial compounds to treat staphylococcal infections. / Funktionelle Membranmikrodomänen (FMMs) in Bakterien sind Membranplattformen, die in strukturellen und funktionellen Aspekten mit Lipid Rafts eukaryotischer Zellen vergleichbar sind. Diese Nanometer-großen, dynamischen Protein-/Lipid-Cluster in der eukaryotischen Zellmembran dienen als Signalzentrum und Assemblierungsplattformen. Allerdings ist die Arbeit an diesen Strukturen durch die Komplexität der eukaryotischen Zellen oft eingeschränkt. Daher sind prokayotische Zellen attraktive Modellsysteme, um molekulare Eigenschaften dieser Art von Membranorganisation zu untersuchen. Ähnlich wie eukaryotische Lipid Rafts, bestehen FMMs aus polyisoprenoiden Lipiden, Scaffold-Proteinen und bestimmten Membranproteinen, die z.B. an Signalweiterleitung und Sekretion beteiligt sind. Die Untersuchung bakterieller FMMs trägt nicht nur dazu bei, die physiologische Relevanz der FMMs in Bakterien selbst zu verstehen, sondern auch um generelle Membranorganisationsprinzipien aufzuklären, die über Bakterien hinausgehen. In dieser Arbeit wurde daher ein bakterieller Modellorganismus benutzt, um Effekte von synthetischer Überproduktion von Raft-assoziierten Scaffold-Proteinen zu untersuchen. Diese Überexpression führt zu einer unüblichen Stabilisierung der Protease FtsH, die in den FMMs zu finden ist, was eine fehlerhafte Regulierung der Zielproteine von FtsH zur Folge hat. Demzufolge sind Entwicklungsdefekte und Anomalien in der Zellform die Konsequenzen, die im Umkehrschluss die Zellphysiologie negativ beeinträchtigen. Diese Ergebnisse können dazu dienen, Lipid-Raft Prozesse in Menschen besser zu verstehen, wo die Hochregulierung von Flotillin im Zusammenhang mit neurologischen Krankheiten steht. Darüber hinaus zielt diese Arbeit darauf ab, das FMM-Proteom des humanen Pathogenes Staphylococcus aureus besser zu verstehen. Eine detaillierte, quantitative Massenspektrometrieanalyse hat ergeben, dass das Proteincargo der FMMs sich zwar verschiedenen Bedingungen anpasst, aber auch ein bestimmtes Kernproteom in allen getesteten Bedingungen beibehält. Als Fallstudie wurde gezeigt, dass die Assemblierung des Typ VII Sekretionssystems von den FMMs, und im Detail von der Aktivität des Scaffoldproteins Flotillin, abhängig ist. Dieses Sekretionssystem ist wichtig für die Virulenzausbildung dieses Pathogenes und die Sekretionseffizienz kann durch kleine Moleküle verringert werden, die die Aktivität von Flotillin inhibieren. Diese Strategie eröffnet neue Möglichkeiten für die Anwendung unkonventioneller, antimikrobieller Substanzen, um Staphylokokken-Infektionen zu behandeln.

München, Techn. Univ., Diss., 2009.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.

The structure and functions of lipid rafts and the mechanisms of intracellular membrane trafficking are major topics in current cell biological research. Rafts have been proposed to act as sorting platforms during biosynthetic transport, especially along pathways that deliver proteins to the apical membrane of polarised cells. Based on this, the aim of this work was to contribute to the understanding of apical sorting in epithelial cells. The study of how lipid rafts are structured has been hampered by the scarcity of techniques for their purification. Rafts are thought to be partially resistant to solubilisation by mild detergents, which has made the isolation of detergent-resistant membranes (DRMs) the primary method to characterise them biochemically. While a growing number of detergents is being used to prepare DRMs, it is not clear what can be inferred about the native structure of cell membranes from the composition of different DRMs. This issue was addressed by an analysis of DRMs prepared with a variety of mild detergents. The protein and lipid content of different DRMs from two cell lines, Madin-Darby canine kidney (MDCK) and Jurkat cells, was compared. It was shown that the detergents differed considerably in their ability to selectively solubilise membrane proteins and lipids. These results make it unlikely that different DRMs reflect the same underlying principle of membrane organisation. Another obstacle for understanding apical sorting is that the evidence implicating certain proteins in this process has come from various disparate approaches. It would be helpful to re-examine the putative components of the apical sorting machinery in a single experimental system. To this end, a retroviral system for RNA interference (RNAi) in MDCK cells was established. Efficient suppression of thirteen genes was achieved by retroviral co-expression of short hairpin RNAs and a selectable marker. In addition, the system was extended to simultaneously target two genes, giving rise to double knockdowns.Retroviral RNAi was applied to deplete proteins implicated in apical sorting. Surprisingly, none of the knockdowns analysed caused defects in surface delivery of influenza virus hemagglutinin, a common marker protein for apical transport. Therefore, none of the proteins examined is absolutely required for transport to the apical membrane of MDCK cells. Cells may adapt to the depletion of proteins involved in membrane trafficking by activating alternative pathways. To avoid such adaptation, a visual transport assay was established. It is based on the adenoviral expression of fluorescent marker proteins whose surface transport can be followed microscopically as soon as RNAi has become effective. With this assay, it should now be possible to screen the knockdowns for defects in surface transport. Taken together, this work has provided a number of experimental tools for the study of membrane trafficking in epithelial cells. First, the biochemical analysis of DRMs highlighted that DRMs obtained with different detergents are unlikely to correspond to distinct types of membrane microdomains in cell membranes. Second, the retroviral RNAi system should be valuable for defining the function of proteins, not only in membrane transport, but also in processes like epithelial polarisation. Third, the visual assay for monitoring the surface transport of adenovirally expressed marker proteins should be suitable to detect defects in polarised sorting.

Eastern Africa has experienced strong climatic changes since the last deglaciation (15 000 years ago). The driving mechanisms and teleconnections of these spatially complex climate variations are yet not fully understood. Although previous studies on lake systems have enhanced our knowledge of Holocene precipitation variation in eastern Africa, relatively few studies have reconstructed the terrestrial temperature history of eastern Africa from lake archives. Here, we present (i) a new branched glycerol dialkyl glycerol tetraether (brGDGT) temperature calibration that includes Bale Mountains surface sediments and (ii) a quantitative record of mean annual air temperature (MAT) over the past 12 ka cal BP using brGDGTs in a sediment core collected from Garba Guracha (3950ma.s.l.) in the Bale Mountains. After adding Bale Mountains surface sediment (n = 11) data (Baxter et al., 2019) to the existing East African lake dataset, additional variation in 6-methyl brGDGTs was observed, which necessitated modifying the MBT'5ME calibration (MBT denotes methylation of branched tetraethers) by adding 6-methyl brGDGT IIIa0 (resulting in the MBT Bale Mountains index, r² = 0:93, p < 0:05). Comparing the MBT'5ME and the new MBT Bale Mountains index, our high-altitude Garba Guracha temperature record shows that warming occurred shortly after the Holocene onset when the temperature increased by more than 3.0°C in less than 600 years. The highest temperatures prevailed between 9 and 6 ka cal BP, followed by a temperature decrease until 1.4 ka cal BP. The reconstructed temperature history is linked to supraregional climatic changes associated with insolation forcing and the African Humid Period (AHP), as well as with local anomalies associated with catchment deglaciation and hydrology.