drp, A novel cell density regulated protein

Growing cells are continuously processing signals of all varieties and responding to these signals by changes in cellular gene expression. One signal that cells in close proximity relay to each other is cell-cell contact. Non-transformed cells respond to cell-cell contact by arrest of growth and entry into G$\sb0,$ a process known as contact inhibition. Transformed cells do not respond to contact inhibition and continue to grow to high cell density, forming foci when in cell culture and tumors in the living organism. The events surrounding the generation, transduction, and response to cellular contact are poorly understood. In the present study, a novel gene product, drp, is shown to be expressed at high levels in cultured cells at high cell density. This density regulated protein, drp, has an apparent molecular weight of 70 kDa. Northern analysis shows drp to be highly expressed in cardiac and skeletal muscle and least abundant in lung and kidney tissues. By homology to two independently derived sequence tagged sites (STSs) used in the human genome project, drp or a closely related sequence maps to human chromosome 12. Density-dependent increases in drp expression have been demonstrated in six different cell lines including NIH 3T3, Hela and a human teratocarcinoma cell line, PA-1. Cells exhibit increased drp expression both when they are plated at increasing concentrations per unit area, or plated at low density and allowed to grow naturally to higher cell density. Cells at high density can exhibit several phenotypes including growth arrest, accumulation of soluble factors in the media, and increased numbers of cell contacts. Growth arrest by serum starvation or TGF-$\beta$ treatment fails to produce an increase in drp expression. Similarly, treatment of low density cells with conditioned media from high density cells fails to elicit drp expression. These results argue that neither soluble factors accumulated or expressed at high density nor simple exit from the cell cycle is sufficient to produce an increase in drp expression. The expression of drp appears to be uniquely regulated by cell density alone. ^

Phototaxis signaling by the membrane complex of archaeal sensory rhodopsins and their transducers

Sensory rhodopsins I and II (SRI and SRII) are visual pigment-like phototaxis receptors in the archaeon Halobacterium salinarum. The receptor proteins each consist of a single polypeptide that folds into 7 $\alpha$-helical membrane-spanning segments forming an internal pocket where the chromophore retinal is bound. They transmit signals to their tightly bound transducer proteins, HtrI and HtrII, respectively, which in turn control a phosphotransfer pathway modulating the flagellar motors. SRI-HtrI mediates attractant responses to orange-light and repellent responses to UV light, while SRII-HtrII mediates repellent response to blue light. Experiments were designed to analyze the molecular processes in the SR-Htr complexes responsible for receptor activation, which previously had been shown by our laboratory to involve proton transfer reactions of the retinylidene Schiff base in the photoactive site, transfer of signals from receptor to transducer, and signaling specificity by the receptor-transducer complex.^ Site-directed mutagenesis and laser-flash kinetic spectroscopy revealed that His-166 in SRI (i) plays a role in the proton transfers both to and from the Schiffbase, either as a structurally critical residue or possibly as a direct participant, (ii) is involved in the modulation of SIU photoreaction kinetics by HtrI, and (iii) modulates the pKa of Asp-76, an important residue in the photoactive site, through a long-distance electrostatic interaction. Computerized cell tracking and motion analysis demonstrated that (iv) His-166 is crucial in phototaxis signaling: a spectrum of substitutions either eliminate signaling or greatly perturb the activation process that produces attractant and repellent signaling states of the receptor.^ The signaling states of SRI are communicated to HtrI, whose oligomeric structure and conformational changes were investigated by engineered sulfhydryl probes. It was found that signaling by the SRI-HtrI complex involves reversible conformational changes within a preexisting HtrI dimer, which is likely accomplished through a slight winding or unwinding of the two HtrT monomers via their loose coiled coil association. To elucidate which domains of the Htr dimers confer specificity for interaction with SRI or SRII, chimeras of HtrI and HtrII were constructed. The only determinant needed for functional and specific interaction with SRI or SRII was found to be the four transmembrane segments of the HtrI or HtrII dimers, respectively. The entire cytoplasmic parts of HtrI and HtrII, which include the functionally important signaling and adaptation domains, were interchangeable.^ These observations support a model in which SRI and SRII undergo conformational changes coupled to light-induced proton transfers in their photoactive sites, and that lateral helix-helix interactions with their cognate transducers' 4-helix bundle in the membrane relay these conformational changes into different states of the Htr proteins which regulate the down-stream phosphotransfer pathway. ^

Protein-protein interaction between phototaxis receptor sensory Rhodopsin I and its transducer HtrI

The molecular complex containing the seven transmembrane helix photoreceptor S&barbelow;ensory R&barbelow;hodopsin I&barbelow; (SRI) and transducer protein HtrI (H&barbelow;alobacterial Transducer for SRI&barbelow;) mediates color-sensitive phototaxis responses in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light (orange + UV light) a repellent response by a two-photon reaction. Three aspects of SRI-HtrI structure/function and the signal transduction pathway were explored. First, the coupling of HtrI to the photoactive site of SRI was analyzed by mutagenesis and kinetic spectroscopy. Second, SRI-HtrI mutations and suppressors were selected and characterized to elucidate the color-sensing mechanism. Third, the signal relay through the transducer-bound histidine kinase was analyzed using an in vitro reconstitution system with known and newly identified taxis components. ^ Twenty-one mutations on HtrI were introduced by site-directed mutagenesis. Several replacements of charged residues perturbed the photochemical kinetics of SRI which led to the finding of a cluster of residues at the membrane/cytoplasm interface in HtrI electrostatically coupled to the photoactive site of SRI. We found by laser-flash kinetic spectroscopy that the transducer and these residues have specific effects on the light-induced proton transfer between the retinal chromophore and the protein. ^ One of the mutations showed an unusual mutant phenotype we called “inverted” signaling, in which the cell produces a repellent response to normally attractant light. Therefore, this mutant (E56Q of HtrI) had lost the color-discrimination by the SRI-HtrI complex. We used suppressor analysis to better understand the phenotype. Certain suppressors resulted in return of attractant responses to orange light but with inversion of the normally repellent response to white light to an attractant response. To explain this and other results, we formulated the Conformational Shuttling model in which the HtrI-SRI complex is poised in a metastable equilibrium of two conformations shifted in opposite directions by orange and white light. We tested this model by behavioral analysis (computerized cell tracking and motion study) of double mutants of inverting and suppressing mutations and the results confirmed the equilibrium-shift explanation. ^ We developed an in vitro system for measuring the effect of purified transducer on the histidine-kinase CheAH that controls the flagellar motor switch. The rate of kinase autophosphorylation was stimulated >2 fold in the reconstitution of the complete signal transduction system from purified components from H. salinarum. The in vitro assay also showed that the kinase activity was reduced in the absence and in the presence of high levels of linker protein CheWH. (Abstract shortened by UMI.) ^

Genome-wide analysis of genetic and epigenetic control of programmed DNA deletion

During the development of the somatic genome from the Paramecium germline genome the bulk of the copies of ∼45 000 unique, internal eliminated sequences (IESs) are deleted. IES targeting is facilitated by two small RNA (sRNA) classes: scnRNAs, which relay epigenetic information from the parental nucleus to the developing nucleus, and iesRNAs, which are produced and used in the developing nucleus. Why only certain IESs require sRNAs for their removal has been enigmatic. By analyzing the silencing effects of three genes: PGM (responsible for DNA excision), DCL2/3 (scnRNA production) and DCL5 (iesRNA production), we identify key properties required for IES elimination. Based on these results, we propose that, depending on the exact combination of their lengths and end bases, some IESs are less efficiently recognized or excised and have a greater requirement for targeting by scnRNAs and iesRNAs. We suggest that the variation in IES retention following silencing of DCL2/3 is not primarily due to scnRNA density, which is comparatively uniform relative to IES retention, but rather the genetic properties of IESs. Taken together, our analyses demonstrate that in Paramecium the underlying genetic properties of developmentally deleted DNA sequences are essential in determining the sensitivity of these sequences to epigenetic control.

Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Atomic mutagenesis of the ribosomal Sarcin-Ricin-Loop to study EF-G GTPase activation

Translocation factor EF-G, possesses a low basal GTPase activity, which is stimulated by the ribosome. One potential region of the ribosome that triggers GTPase activity of EF-G is the Sarcin-Ricin-Loop (SRL) (helix 95) in domain VI of the 23S rRNA. Structural data showed that the tip of the SRL closely approaches GTP in the active center of EF-G, structural probing data confirmed that EF-G interacts with nucleotides G2655, A2660, G2661 and A2662.1-3 The exocyclic group of adenine at A2660 is required for stimulation of EF-G GTPase activity by the ribosome as demonstrated using atomic mutagenesis.4 Recent crystal structures of EF-G on the ribosome, gave more insights into the molecular mechanism of EF-G GTPase activity.5 Based on the structure of EF-Tu on the ribosome1, the following mechanism of GTPase activation was proposed: upon binding of EF-G to the ribosome, the conserved His92 (E.coli) changes its position, pointing to the γ-phosphate of GTP. In this activated state, the phosphate of residue A2662 of the SRL positions the catalytic His in its active conformation. It was further proposed that the phosphate oxygen of A2662 is involved in a charge-relay system, enabling GTP hydrolysis. In order to test this mechanism, we use the atomic mutagenesis approach, which allows introducing non-natural modifications in the SRL, in the context of the complete 70S ribosome. Therefore, we replaced one of the non-bridging oxygens of A2662 by a methyl group. A methylphosphonat is not able to position or activate a histidine, as it has no free electrons and therefore no proton acceptor function. These modified ribosomes were then tested for stimulation of EF-G GTPase activity. First experiments show that one of the two stereoisomers incorporated into ribosomes does not stimulate GTPase activity of EF-G, whereas the other is active. From this we conclude that indeed the non-bridging phosphate oxygen of A2662 is involved in EF-G GTPase activation by the ribosome. Ongoing experiments aim at revealing the contribution of this non-bridging oxygen at A2662 to the mechanism of EF-G GTPase activation at the atomic level.

Optical Tolerance Analysis of the Multi-Beam Limb Viewing Instrument STEAMR

In this paper, we report on an optical tolerance analysis of the submillimeter atmospheric multi-beam limb sounder, STEAMR. Physical optics and ray-tracing methods were used to quantify and separate errors in beam pointing and distortion due to reflector misalignment and primary reflector surface deformations. Simulations were performed concurrently with the manufacturing of a multi-beam demonstrator of the relay optical system which shapes and images the beams to their corresponding receiver feed horns. Results from Monte Carlo simulations show that the inserts used for reflector mounting should be positioned with an overall accuracy better than 100 μm (~ 1/10 wavelength). Analyses of primary reflector surface deformations show that a deviation of magnitude 100 μm can be tolerable before deployment, whereas the corresponding variations should be less than 30 μm during operation. The most sensitive optical elements in terms of misalignments are found near the focal plane. This localized sensitivity is attributed to the off-axis nature of the beams at this location. Post-assembly mechanical measurements of the reflectors in the demonstrator show that alignment better than 50 μm could be obtained.

Finding a voice - Nollywood inspired filmmaking practices in Switzerland

Under the name Nollywood a unique video film industry has developed in Nigeria in the last few decades, which now forms one of the world’s biggest entertainment industries. With its focus on stories reflecting „the values, desires and fears” (Haynes 2007: 133) of African viewers and its particular way of production, Nollywood brings „lived practices and its representation together in ways that make the films deeply accessible and entirely familiar to their audience“ (Marston et al. 2007: 57). In doing so, Nollywood shows its spectators new postcolonial forms of performative self‐expression and becomes a point of reference for a wide range of people. However, Nollywood not only excites a large number of viewers inside and outside Nigeria, it also inspires some of them to become active themselves and make their own films. This effect of Nigerian filmmaking can be found in many parts of sub‐Saharan Africa as well as in African diasporas all over the world – including Switzerland (Mooser 2011: 63‐66). As a source of inspiration, Nollywood and its unconventional ways of filmmaking offer African migrants a benchmark that meets their wish to express themselves as minority group in a foreign country. As Appadurai (1996: 53), Ginsburg (2003: 78) and Marks (2000: 21) assume, filmmakers with a migratory background have a specific need to express themselves through media. As minority group members in their country of residence they not only wish to reflect upon their situation within the diaspora and illustrate their everyday struggles as foreigners, but to also express their own views and ideas in order to challenge dominant public opinion (Ginsburg 2003: 78). They attempt to “talk back to the structures of power” (2003: 78) they live in. In this process, their audio-visual works become a means of response and “an answering echo to a previous presentation or representation” (Mitchell 1994: 421). The American art historian Mitchell, therefore, suggests interpreting representation as “the relay mechanism in exchange of power, value, and publicity” (1994: 420). This desire of interacting with the local public has also been expressed during a film project of African, mainly Nigerian, first-generation migrants in Switzerland I am currently partnering in. Several cast and crew members have expressed feelings of being under-represented, even misrepresented, in the dominant Swiss media discourse. In order to create a form of exchange and give themselves a voice, they consequently produce a Nollywood inspired film and wish to present it to the society they live in. My partnership in this on‐going film production (which forms the foundation of my PhD field study) allows me to observe and experience this process. By employing qualitative media anthropological methods and in particular Performance Ethnography, I seek to find out more about the ways African migrants represent themselves as a community through audio‐visual media and the effect the transnational use of Nollywood has on their form of self‐representations as well as the ways they express themselves.

The Osr1 and Osr2 genes act in the pronephric anlage downstream of retinoic acid signaling and upstream of Wnt2b to maintain pectoral fin development.

Vertebrate odd-skipped related genes (Osr) have an essential function during the formation of the intermediate mesoderm (IM) and the kidney structures derived from it. Here, we show that these genes are also crucial for limb bud formation in the adjacent lateral plate mesoderm (LPM). Reduction of zebrafish Osr function impairs fin development by the failure of tbx5a maintenance in the developing pectoral fin bud. Osr morphant embryos show reduced wnt2b expression, and increasing Wnt signaling in Osr morphant embryos partially rescues tbx5a expression. Thus, Osr genes control limb bud development in a non-cell-autonomous manner, probably through the activation of Wnt2b. Finally, we demonstrate that Osr genes are downstream targets of retinoic acid (RA) signaling. Therefore, Osr genes act as a relay within the genetic cascade of fin bud formation: by controlling the expression of the signaling molecule Wnt2ba in the IM they play an essential function transmitting the RA signaling originated in the somites to the LPM.

Information-centric communication in mobile and wireless networks

Information-centric networking (ICN) is a new communication paradigm that has been proposed to cope with drawbacks of host-based communication protocols, namely scalability and security. In this thesis, we base our work on Named Data Networking (NDN), which is a popular ICN architecture, and investigate NDN in the context of wireless and mobile ad hoc networks. In a first part, we focus on NDN efficiency (and potential improvements) in wireless environments by investigating NDN in wireless one-hop communication, i.e., without any routing protocols. A basic requirement to initiate informationcentric communication is the knowledge of existing and available content names. Therefore, we develop three opportunistic content discovery algorithms and evaluate them in diverse scenarios for different node densities and content distributions. After content names are known, requesters can retrieve content opportunistically from any neighbor node that provides the content. However, in case of short contact times to content sources, content retrieval may be disrupted. Therefore, we develop a requester application that keeps meta information of disrupted content retrievals and enables resume operations when a new content source has been found. Besides message efficiency, we also evaluate power consumption of information-centric broadcast and unicast communication. Based on our findings, we develop two mechanisms to increase efficiency of information-centric wireless one-hop communication. The first approach called Dynamic Unicast (DU) avoids broadcast communication whenever possible since broadcast transmissions result in more duplicate Data transmissions, lower data rates and higher energy consumption on mobile nodes, which are not interested in overheard Data, compared to unicast communication. Hence, DU uses broadcast communication only until a content source has been found and then retrieves content directly via unicast from the same source. The second approach called RC-NDN targets efficiency of wireless broadcast communication by reducing the number of duplicate Data transmissions. In particular, RC-NDN is a Data encoding scheme for content sources that increases diversity in wireless broadcast transmissions such that multiple concurrent requesters can profit from each others’ (overheard) message transmissions. If requesters and content sources are not in one-hop distance to each other, requests need to be forwarded via multi-hop routing. Therefore, in a second part of this thesis, we investigate information-centric wireless multi-hop communication. First, we consider multi-hop broadcast communication in the context of rather static community networks. We introduce the concept of preferred forwarders, which relay Interest messages slightly faster than non-preferred forwarders to reduce redundant duplicate message transmissions. While this approach works well in static networks, the performance may degrade in mobile networks if preferred forwarders may regularly move away. Thus, to enable routing in mobile ad hoc networks, we extend DU for multi-hop communication. Compared to one-hop communication, multi-hop DU requires efficient path update mechanisms (since multi-hop paths may expire quickly) and new forwarding strategies to maintain NDN benefits (request aggregation and caching) such that only a few messages need to be transmitted over the entire end-to-end path even in case of multiple concurrent requesters. To perform quick retransmission in case of collisions or other transmission errors, we implement and evaluate retransmission timers from related work and compare them to CCNTimer, which is a new algorithm that enables shorter content retrieval times in information-centric wireless multi-hop communication. Yet, in case of intermittent connectivity between requesters and content sources, multi-hop routing protocols may not work because they require continuous end-to-end paths. Therefore, we present agent-based content retrieval (ACR) for delay-tolerant networks. In ACR, requester nodes can delegate content retrieval to mobile agent nodes, which move closer to content sources, can retrieve content and return it to requesters. Thus, ACR exploits the mobility of agent nodes to retrieve content from remote locations. To enable delay-tolerant communication via agents, retrieved content needs to be stored persistently such that requesters can verify its authenticity via original publisher signatures. To achieve this, we develop a persistent caching concept that maintains received popular content in repositories and deletes unpopular content if free space is required. Since our persistent caching concept can complement regular short-term caching in the content store, it can also be used for network caching to store popular delay-tolerant content at edge routers (to reduce network traffic and improve network performance) while real-time traffic can still be maintained and served from the content store.

Nota crítica a Demóstenes 19.94

La interpretación y el texto mismo de unas líneas del parágrafo 94 del discurso Sobre la embajada fraudulenta de Demóstenes han sido objeto de discusiones, en particular la expresión ten proten eferon tu fenakismu. En nuestra opinión, el problema se podría resolver si interpretamos que a lo largo de todo el pasaje se desarrolla una metáfora deportiva, en concreto relacionada con las lampadedromiai o carreras de relevos con antorchas

Dive depth and dive duration data of subadult and adult, male and female southern elephant seals from Marion Island between 2004 and 2008 with links to datasets

Although numerous studies have addressed the migration and dive behaviour of southern elephant seals (Mirounga leonina), questions remain about their habitat use in the marine environment. We report on the vertical use of the water column in the species and the potential lifetime implications for southern elephant seals from Marion Island. Long-term mark-resight data were used to complement vertical habitat use for 35 known individuals tagged with satellite-relay data loggers, resulting in cumulative depth use extrapolated for each individual over its estimated lifespan. Seals spent on average 77.59% of their lives diving at sea, 7.06% at the sea surface, and 15.35% hauled out on land. Some segregation was observed in maximum dive depths and depth use between male and female animals-males evidently being physiologically more capable of exploiting increased depths. Females and males spent 86.98 and 80.89% of their lives at sea, respectively. While at sea, all animals spent more time between 300 and 400 m depth, than any other depth category. Males and females spent comparable percentages of their lifetimes below 100 m depth (males: 65.54%; females: 68.92%), though males spent 8.98% of their lives at depths in excess of 700 m, compared to females' 1.84% at such depths. Adult males often performed benthic dives in excess of 2,000 m, including the deepest known recorded dive of any air-breathing vertebrate (>2,133 m). Our results provide a close approximation of vertical habitat use by southern elephant seals, extrapolated over their lifespans, and we discuss some physiological and developmental implications of their variable depth use.

Nota crítica a Demóstenes 19.94

La interpretación y el texto mismo de unas líneas del parágrafo 94 del discurso Sobre la embajada fraudulenta de Demóstenes han sido objeto de discusiones, en particular la expresión ten proten eferon tu fenakismu. En nuestra opinión, el problema se podría resolver si interpretamos que a lo largo de todo el pasaje se desarrolla una metáfora deportiva, en concreto relacionada con las lampadedromiai o carreras de relevos con antorchas

Nota crítica a Demóstenes 19.94

La interpretación y el texto mismo de unas líneas del parágrafo 94 del discurso Sobre la embajada fraudulenta de Demóstenes han sido objeto de discusiones, en particular la expresión ten proten eferon tu fenakismu. En nuestra opinión, el problema se podría resolver si interpretamos que a lo largo de todo el pasaje se desarrolla una metáfora deportiva, en concreto relacionada con las lampadedromiai o carreras de relevos con antorchas

Real time power plant simulation platform for training on Electrical Protections and Automatic Voltage Regulators

Electrical Protection systems and Automatic Voltage Regulators (AVR) are essential components of actual power plants. Its installation and setting is performed during the commissioning, and it needs extensive experience since any failure in this process or in the setting, may entails some risk not only for the generator of the power plant, but also for the reliability of the power grid. In this paper, a real time power plant simulation platform is presented as a tool for improving the training and learning process on electrical protections and automatic voltage regulators. The activities of the commissioning procedure which can be practiced are described, and the applicability of this tool for improving the comprehension of this important part of the power plants is discussed. A commercial AVR and a multifunction protective relay have been tested with satisfactory results.