Control of echolocation pulses by neurons of the nucleus ambiguus in the rufous horseshoe bat, Rhinolophus rouxi

1. Horseradish peroxidase was applied by iontophoretic injections to physiologically identified regions of the laryngeal motor nucleus, the nucleus ambiguus in the CF/FM batRhinolophus rouxi. 2. The connections of the nucleus ambiguus were analysed with regards to their possible functional significance in the vocal control system, in the respiration control system, and in mediating information from the central auditory system. 3. The nucleus ambiguus is reciprocally interconnected with nuclei involved in the generation of the vocal motor pattern, i.e., the homonomous contralateral nucleus and the area of the lateral reticular formation. Similarly, reciprocal connections are found with the nuclei controlling the rhythm of respiration, i.e., medial parts of the medulla oblongata and the parabrachial nuclei. 4. Afferents to the nucleus ambiguus derive from nuclei of the descending vocalization system (periaqueductal gray and cuneiform nuclei) and from motor control centers (red nucleus and frontal cortex). 5. Afferents to the nucleus ambiguus, possibly mediating auditory influence to the motor control of vocalization, come from the superior colliculus and from the pontine nuclei. The efferents from the pontine nuclei are restricted to rostral parts of the nucleus ambiguus, which hosts the motoneurons of the cricothyroid muscle controlling the call frequency

Laryngeal Nerve Activity During Pulse Emission in the CF-FM Bat, Rhinolophus ferrumequinum. II. The Recurrent Laryngeal Nerve

The activity of the recurrent laryngeal nerve (RLN) was recorded in the greater horseshoe bat,Rhinolophus ferrumequinum. Respiration, vocalization and nerve discharges were monitored while vocalizations were elicted by stimulation of the central gray matter. This stimulation evoked either expiration or expiration plus vocalization depending on the stimulus strength. When vocalization occurred it always took place during expiration. Recordings from the RLN during respiration showed activity during the inspiration phase, but when vocalization occurred there was activity during inspiration and expiration. These results are consistent with the view that the RLN innervates muscles which control the opening and closing of the glottis. During vocalization the vocal folds are closely approximated and the discharge patterns of the nerve suggests that it controls the muscles which start and end each pulse

Identification of translationally deregulated proteins during inflammation-associated tumorigenesis

The translation of mRNAs into proteins is an elaborate and highly regulated process. Translational regulation primarily takes place at the level of initiation. During initation the eukaryotic initiation factors (eIFs) form a complex that binds to the 5’end of the mRNA to scan for a start codon. Once recognized, the ribosome is recruited to the mRNA and protein synthesis starts. Initiation of translation can basically occur via two distinct mechanisms, i.e. cap-dependent and cap-independent that is mediated via internal ribosome entry sites (IRESs). The former is mediated by a 5’cap structure composed of a 7-methylguanylate which is added to every mRNA during transcription and recruits the initiation complex. IRES-dependent translation involves elements within the 5’untranslated region (UTR) of the mRNA that mostly bind IRES trans-acting factors (ITAFs) which associate either with the initiation complex or with the ribosome itself and consequently allow for internal initiation of translation. During tumorigenesis the demand for proteins is increased due to rapid cell growth, which consequently requires enhanced translation. Many factors that regulate translation are overexpressed in tumors. Moreover, signaling pathways that trigger translation or further hyperactivated by the surrounding tumor microenvironment. This environment is largely generated by infiltration of immune cells such as macrophages that secrete cytokines and other mediators to promote tumorigenesis. As the effects of inflammatory conditions on the translation of specific targets are only poorly characterized, my study aimed at identifying translationally deregulated targets during inflammation-associated tumorigenesis. For this purpose, I cocultured MCF7 breast tumor cells with conditioned medium of activated monocyte-derived U937 macrophages (CM). Polysome profiling and microarray analysis identified 42 targets to be regulated at the level of translation. The results were validated by quantitative PCR and one target - early growth response 2 (EGR2) - was chosen for in depth analysis of the mechanism leading to its enhanced translation. In order to identify upstream signaling molecules causing enhanced EGR2 protein synthesis the cytokine profile of CM was analyzed and the impact of several cytokines on EGR2 translation was examined. Preincubation of CM with neutralizing antibodies revealed that lowering interleukin 6 (IL-6) had only little effect, whereas depletion of IL 1β significantly reduced EGR2 translation. This finding was corroborated by the fact that treatment with recombinant IL-1β enhanced EGR2 translation to virtually the same extend as CM. Further experiments revealed that this effect was mediated via the p38-MAPK signaling cascade. Interestingly, I observed that the mTOR inhibitor rapamycin, which reduces cap-dependent translation, specifically stimulated EGR2 translation. This result argued for an IRES-dependent mechanism that might account for EGR2 translation. The use of bicistronic reporter assays verified this hypothesis. In line with the above mentioned results, CM, IL-1β and p38-MAPK induced EGR2-IRES activity. Since IRESs commonly require ITAFs to mediate translation initiation, the binding of proteins to the 5’UTR was analyzed using mass spectrometry. Among others, several previously described ITAFs, such as polypyrimidine tract-binding protein (PTB) and heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) were identified to directly bind to the EGR2-5’UTR. Furthermore, overexpression of hnRNP-A1 enhanced EGR2-IRES activity whereas a dominant negative form of hnRNP-A1 significantly decreased it, thus, showing its importance for EGR2 translation. In summary, my data provide evidence that EGR2 expression can be controlled by IRES-dependent translational regulation, which is responsive to an inflammatory environment. The identified mechanism may not be exclusive for one target but might be representative for gene expression regulation mechanisms during tumorigenesis. This is of special interest for the treatment of cancer patients and development of more specific therapies to reduce tumor outcome.Die Translation von mRNAs in Proteine ist ein komplexer Prozess, der aufgrund seines hohen Energieverbrauchs strikt kontrolliert wird. Die Regulation findet dabei primär auf Ebene der Translationsinitiation statt. Während der Initiation bilden die eukaryotischen Initiationsfaktoren (eIFs) einen Komplex, der an das 5’Ende der mRNA bindet und die 5’untranslatierte Region (UTR) nach einem Startcodon scannt, woraufhin die ribosomalen Untereinheiten an die mRNA rekrutiert werden. Die Ribosomen vermitteln dann die eigentliche Proteinsynthese. Grundsätzlich können zwei verschiedene Arten der Initiation unterschieden werden – die Cap-abhängige sowie die Cap-unabhängige, wobei letztere über sogenannte internal ribosome entry sites (IRESs) vermittelt wird. Bei ersterer bindet der Initiationskomplex an die Cap-Struktur der mRNA, die aus einem N-terminalen 7 Methylguanylat besteht. Bei der IRES-vermittelten Initiation bindet der Initiationskomplex oder auch die kleine ribosomale Untereinheit direkt innerhalb der 5’UTR an die mRNA, allerdings in 3’-Distanz zur Cap-Struktur. Während der Tumorentwicklung kommt es aufgrund des verstärkten Zellwachstums zu einem gesteigerten Bedarf an Proteinen und somit zu erhöhter Translation. Viele Faktoren, die die Translation regulieren, werden in Tumoren überexprimiert oder sind überaktiv. Bei der Aktivierung der entsprechenden Signalkaskaden spielt das Tumormilieu eine zentrale Rolle. Dieses wird insbesondere von Zellen des Immunsystems wie z.B. Makrophagen beeinflusst. Makrophagen setzen dabei Mediatoren frei, welche das Tumorwachstum begünstigen. Während tumorigene Expressionsveränderungen auf Transkriptionsebene bereits detailliert untersucht wurden, gibt es nur wenig Information über Translationsveränderungen spezifischer Proteine. Deswegen war es das Ziel dieser Studie translationell (de-)regulierte Proteine in der entzündungsinduzierten Tumorigenese zu identifizieren. Dafür kokultivierte ich MCF7 Brustkrebszellen mit konditioniertem Medium von ausdifferenzierten U937 Makrophagen (CM). Die Translationsveränderung in den Tumorzellen wurde mit Hilfe von Polysomenfraktionierungen überprüft. Durch eine aufbauende Mikroarray Analyse wurden 42 mRNAs identifiziert, die translationell reguliert wurden. Die Ergebnisse des Mikroarrays wurden anschließend durch quantitative PCR validiert und der Regulationsmechanismus eines Targets – early growth response 2 (EGR2) – im Detail analysiert. Dafür untersuchte ich den Einfluss verschiedener im CM vorhandener Zytokine auf die EGR2-Translation mittels neutralisierender Antikörper. Es stellte sich heraus, dass die Abreicherung von Interleukin 6 (IL-6) die EGR2-Translationsinduktion durch CM nur minimal verringerte, wohingegen eine Depletion von IL-1β diese signifikant inhibierte. Dieser Befund wurde dadurch unterstützt, dass eine Behandlung mit rekombinantem IL-1β eine ähnlich starke Induktion der EGR2-Translation bewirkte wie CM. Anschließende Untersuchungen ergaben, dass dieser Effekt durch die p38-MAPK Signalkaskade vermittelt wurde. Desweiteren wurde beobachtet, dass der mTOR-Inhibitor Rapamycin, der die Cap-abhängige Translation hemmt, ebenfalls zu einer verstärkten EGR2-Translation führte. Dies ließ vermuten, dass ein IRES-vermittelter Mechanismus der Translation von EGR2 zu Grunde lag. Durch die Verwendung von bicistronischen Reporter-Vektoren wurde diese Hypothese bestätigt. Außerdem konnte ich beweisen, dass CM, IL 1β und p38-MAPK die EGR2-IRES-Aktivität in der gleichen Art beeinflussten wie bereits für die EGR2-Translation mittels Polysomenfraktionierung gezeigt. Da zelluläre IRES-Elemente oft durch sogenannte IRES trans-acting factors (ITAFs) induziert werden, wurden mittels Massenspektrometrie Proteine identifiziert, die an die 5’UTR von EGR2 binden. Unter anderem wurden die bereits bekannten ITAFs polypyrimidine tract-binding protein (PTB) und heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) gefunden. Abschließend konnte bewiesen werden, dass die Überexpression von hnRNP-A1 zu einer Erhöhung der EGR2-IRES-Aktivität führte, wohingegen eine dominant-negative Mutante von hnRNP-A1 diese signifikant inhibierte. Diese Ergebnisse ließen darauf schließen, dass hnRNP-A1 einen entscheidenden Einfluss auf die IRES-abhängige EGR2-Translation hat. Zusammenfassend konnte ich einen neuen Translationsregulationsmechanismus für EGR2 identifizieren, der durch ein entzündliches Tumormikroenvironment in Tumorzellen induziert wird. Dieser Mechanismus ist möglicherweise auch auf weitere translationell regulierte Targets übertragbar. Dies ist von besonderem Interesse, da es für eine optimale Behandlung von Tumorpatienten essentiell ist die zu Grunde liegenden Regulationsmechanismen zu verstehen

Is it tonotopy after all?

In this functional MRI study the frequency-dependent localization of acoustically evoked BOLD responses within the human auditory cortex was investigated. A blocked design was employed, consisting of periods of tonal stimulation (random frequency modulations with center frequencies 0.25, 0.5, 4.0, and 8.0 kHz) and resting periods during which only the ambient scanner noise was audible. Multiple frequency-dependent activation sites were reliably demonstrated on the surface of the auditory cortex. The individual gyral pattern of the superior temporal plane (STP), especially the anatomy of Heschl's gyrus (HG), was found to be the major source of interindividual variability. Considering this variability by tracking the frequency responsiveness to the four stimulus frequencies along individual Heschl's gyri yielded medio-lateral gradients of responsiveness to high frequencies medially and low frequencies laterally. It is, however, argued that with regard to the results of electrophysiological and cytoarchitectonical studies in humans and in nonhuman primates, the multiple frequency-dependent activation sites found in the present study as well as in other recent fMRI investigations are no direct indication of tonotopic organization of cytoarchitectonical areas. An alternative interpretation is that the activation sites correspond to different cortical fields, the topological organization of which cannot be resolved with the current spatial resolution of fMRI. In this notion, the detected frequency selectivity of different cortical areas arises from an excess of neurons engaged in the processing of different acoustic features, which are associated with different frequency bands. Differences in the response properties of medial compared to lateral and frontal compared to occipital portions of HG strongly support this notion

Verborgen, vergessen, verloren? Perspektiven der Quellenerschließung durch die digitalen "Regesta Imperii"

Bei Projekten zur Digitalisierung in Geisteswissenschaften ist heute die Realisierung größerer, überregionaler und über das World Wide Web abfragbarer Lösungen erforderlich. Die Beiträge dieses Bandes wurden auf der Tagung des Staatsarchivs Hamburg und des Zentrums "Geisteswissenschaften in der digitalen Welt" an der Universität Hamburg am 10. und 11. April 2006 gehalten. Sie leisten einen interdisziplinären Beitrag zur erforderlichen Standardisierung dieser Angebote, die erst den dringend notwendigen Austausch erleichtern und die gemeinsame Nutzung strukturierter Daten ermöglichen kann.Today, digitization projects in the Humanities require the implementation of larger, supraregional solutions. The contributions in this volume were presented at the conference of the Hamburg State Archives and the Center for the Humanities in the Digital World at the University of Hamburg on April 10 and 11, 2006. They make an interdisciplinary contribution to the required standardisation of corresponding services which can only facilitate the urgently needed exchange of information and make it possible to share structured data

Evidence-based Accountability Audits for Cloud Computing

Cloud computing is known for its on-demand service provisioning and has now become mainstream. Many businesses as well as individuals are using cloud services on a daily basis. There is a big variety of services that ranges from the provision of computing resources to services such as productivity suites and social networks. The nature of these services varies heavily in terms of what kind of information is being out-sourced to the cloud provider. Often, that data is sensitive, for instance when PII is being shared by an individual. Also, businesses that move (parts of) their processes to the cloud are actively participating in a major paradigm shift from having data on-premise to transfering data to a third-party provider. However, many new challenges come along with this trend, which are closely tied to the loss of control over data. When moving to the cloud, direct control over geographical storage location, who has access to it and how it is shared and processed is given up. Because of this loss of control, cloud customers have to trust cloud providers that they treat their data in an appropriate and responsible way. Cloud audits can be used to check how data has been processed in the cloud (i.e., by whom, for what purpose) and whether or not this happened in compliance with what has been defined in agreed-upon privacy and data storage, usage and maintenance (i.e., data handling) policies. This way, a cloud customer can regain some of the control he has given up by moving to the cloud. In this thesis, accountability audits are presented as a way to strengthen trust in cloud computing by providing assurance about the processing of data in the cloud according to data handling and privacy policies. In cloud accountability audits, various distributed evidence sources need to be considered. The research presented in this thesis discusses the use of various heterogeous evidence sources on all cloud layers. This way, a complete picture of the actual data handling practices that is based on hard facts can be presented to the cloud consumer. Furthermore, this strengthens transparency of data processing in the cloud, which can lead to improved trust in cloud providers, if they choose to adopt these mechanisms in order to assure their customers that their data is being handled according to their expectations. The system presented in this thesis enables continuous auditing of a cloud provider's adherence to data handling policies in an automated way that shortens audit intervals and that is based on evidence that is produced by cloud subsystems. An important aspect of many cloud offerings is the combination of multiple distinct cloud services that are offered by independent providers. Data is thereby freuqently exchanged between the cloud providers. This also includes trans-border flows of data, where one provider may be required to adhere to more strict data protection requirements than the others. The system presented in this thesis addresses such scenarios by enabling the collection of evidence at providers and evaluating it during audits. Securing evidence quickly becomes a challenge in the system design, when information that is needed for the audit is deemed sensitive or confidential. This means that securing the evidence at-rest as well as in-transit is of utmost importance, in order not to introduce a new liability by building an insecure data heap. This research presents the identification of security and privacy protection requirements alongside proposed solutions that enable the development of an architecture for secure, automated, policy-driven and evidence-based accountability audits