Sensory overload is the feeling of over-stimulation that may lead to increased anxiety and panic in individuals with psychiatric disorders such as autism, post traumatic stress disorder, etc. Understanding visual processing is crucial to enhancing our treatments for disorders where sensory overload is a symptom. How do changes in internal states such as stress or hunger alter visual processing? This project aims to explore how visual processing is affected by signaling in the hypothalamus, an area of the brain regulating changes in internal states and stress. Preliminary studies revealed there are a number of neurons projecting from the lateral area of the hypothalamus to the visual cortex. We want to understand the specific location, identity, and neural circuits of these neurons. Visual cortex neurons were retrogradely traced to identify which inputs originate from the hypothalamus, and the geographical location of these cells was mapped out. The molecular identities of these projection neurons was further explored using specific RNAScope probes to check if those cells are expressing any of four genes most commonly expressed in the hypothalamus: Gal, Crh, Hcrt, and Pmch. This exploration will help us understand the type of signals communicated from the hypothalamic nuclei to the visual cortex to modulate visual processing.

No description available.

No description available.

No description available.

No description available.

The survival of several neuron populations during development, including sympathetic neurons, is strictly regulated by neurotrophins such as nerve growth factor (NGF) released from innervation targets. NGF activates its receptor, TrkA, at axon terminals, to generate signals that are transmitted retrogradely to cell bodies to induce signaling cascades regulating survival. A general view of this process is that NGF generates retrograde survival signals that, when delivered to cell bodies, induce downstream survival signaling that prevents apoptosis. A retrograde survival signal proposed to be necessary for sympathetic neuron survival consists of endosomes containing NGF and phosphorylated TrkA. For this signal, phosphorylated TrkA arriving at cell bodies is required to initiate survival signaling. Studies have tested the necessity of TrkA phosphorylation in the cell bodies for survival: results from different studies contradict each other. Moreover, the Trk inhibitor, K252a, used in these studies, has reported non-specific effects. Using an alternate Trk inhibitor, Gö6976, data presented in this thesis demonstrates that NGF can promote survival by retrograde signaling that does not require TrkA phosphorylation in the cell bodies. These retrograde signals may be composed of signaling molecules activated downstream of TrkA in axons since pro-survival molecules downstream of TrkA, Akt and CREB, were found activated in the cell bodies/proximal axons. Data presented in this thesis also reveals a fundamentally different mechanism for how NGF promotes sympathetic neuron survival: a retrograde apoptotic signal that is suppressed by NGF. NGF withdrawal from axons induced the “axon apoptotic signal” that was retrogradely transmitted to cell bodies to activate a key pro-apoptotic molecule, c-jun. The axon apoptotic signal, which was blocked by the kinase inhibitors rottlerin and chelerythrine, was necessary for apoptosis in response to NGF deprivation. Evidence GSK3 is involved in generation or transmission of the axon apoptotic signal was provided by experiments with GSK3 inhibitors and siRNA. The axon apoptotic signal discovery refutes the previous view that NGF acting on axon terminals supports survival exclusively by generating retrograde survival signals. The axon apoptotic signal has broad implications for understanding nervous system development and other conditions where neuronal apoptosis occurs, such as neurotrauma and neurodegenerative diseases.

L’introduction commence avec la description de toxines d’origines bactérienne et végétale, en particulier la toxine Shiga ainsi que les toxines de la même famille (chapitre 9.1.2). Les petites molécules inhibitrices de ces toxines sont ensuite résumées dans le chapitre 9.1.3, en particulier le composé Retro-2. L’efficacité de ces toxines à atteindre leurs cibles reposant sur le trafic intracellulaire, un aperçu général de l’endocytose et du trafic endosomal sont présentés (chapitre 9.2). Puis, l’entrée de la voie rétrograde est décrite (chapitre 9.2.5), avec un intérêt particulier porté sur la clathrine, le rétromère et GPP130, une protéine qui circule de manière continue entre le Golgi, la membrane plasmique et les endosomes. Les protéines SNARE, en particulier la syntaxine-5 et le syntaxine-16, sont ensuite introduites (chapitre 9.2.6). Après une brève section sur les micro-ARNs de la famille 199 (chapitre 9.3), l’introduction se termine avec la description des techniques clés utilisées au cours de mon travail, tels que la chimie click bio-orthogonale, la synchronisation du trafic antérograde par rétention grâce à des hameçons spécifiques (RUSH), et la ligation par proximité basé sur des anticorps (chapitre 9.4).Ci-inclus, mon article en cours de soumission ouvre la partie résultats (chapitre 10.1), dans laquelle je présente l’intérêt de la chimie click bio-orthogonale pour identifier les cibles cellulaires de Retro-2. Je décris un des candidats potentiels, Sec16A, et illustre comment grâce à la technique de RUSH, perturber la fonction de Sec16A conduit à la relocalisation partielle de la syntaxin-5 au niveau du reticulum endoplasmique via l’inhibition du transport antérograde de la syntaxine-5. La seconde partie de l’article décrit comment la relocalisation de la syntaxine-5 induit l’inhibition du trafic de la toxine Shiga des endosomes au TGN. Je présente une nouvelle interaction entre la syntaxine-5 et la protéine TGN GPP130, qui ont déjà été caractérisées en relation avec le trafic de la toxine Shiga. Mon travail connecte à la fois les facteurs de trafic avec le trafic rétrograde au niveau de l’interface endosome-TGN. De manière frappante, cette interaction est très probablement basée sur une fonction non-SNARE de la syntaxine-5 car le domaine de fixation sur GPP130 est structurellement non lié à toute fonction SNARE.En collaboration avec Juan Francisco Aranda et Carlos Fernandez aux Etats-Unis, nous avons placés des micro-ARNs dans un contexte de régulation endogène du trafic rétrograde de la toxine Shiga (chapitre 11.2). Une discussion plus approfondie sera apportée dans le chapitre 12.Enfin, une vue d’ensemble des projets en cours est apportée dans la section des perspectives (chapitre 12), dans laquelle les collaborations plus approfondies sont mises en lumière.Mots clés : transport rétrograde, toxine Shiga, toxine de la famille Shiga, STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, chimie click sans cuivre, identification des cibles de petites molécules, spétrométrie de masse, function non-SNARE, inhibition du trafic antérograde, miARN, miR199, rétromère, VPS26 / The introduction of my PhD manuscript starts with describing plant and bacterial toxins (chapter 9.1), in particular Shiga toxin and Shiga-like toxins (SLTs) (chapter 9.1.2). Small molecule inhibitors of these toxins are summarized afterwards in chapter 9.1.3, notably the Retro-2 compound. Since these toxins rely on intracellular trafficking to reach their molecular targets, a general overview of endocytosis and endosomal trafficking is provided (chapter 9.2). Next, the retrograde route entry is presented (chapter 9.2.5), with focus on clathrin, the retromer and GPP130, a protein that constantly cycles between Golgi, plasma membrane, and endosomes. SNARE proteins, particularly syntaxin-5 and syntaxin-16, are then introduced (chapter 9.2.6). After a brief section of the micro RNA family 199 (chapter 9.3), the introduction finishes with the description of some salient techniques that were used in my work, such as - bio-orthogonal Click-Chemistry, anterograde trafficking synchronization with the retention using selective hooks (RUSH) assay, and the antibody-based proximity ligation assay (chapter 10.6.1, 0, 10.11.1).Herein, my submitted publication opens the results part (chapter 11.1), in which I present the utility of biorthogonal click chemistry for the search of the cellular targets of Retro-2, a small molecule inhibitor that was previously shown to protect cells and animals against Shiga toxin and ricin. I describe that Sec16A is a likely cellular target candidate, and illustrate using the RUSH approach how interfering with Sec16A functions leads to the partial relocalization of syntaxin-5 to the endoplasmic reticulum (ER) by slowing-down its anterograde transport. The second part of the paper describes how syntaxin-5 relocalization causes the inhibition of Shiga toxin trafficking from endosomes to the TGN. I present a novel interaction between syntaxin-5 and the Golgi protein GPP130, which both have been already described in relation to Shiga toxin trafficking. My work connects both trafficking factors in retrograde trafficking at the endosomes-TGN interface. Strikingly, I demonstrate that this interaction is most probably based on a non-SNARE function of syntaxin-5.In collaboration with Juan Francisco Aranda and Carlos Fernandez in the US, we put micro RNAs into an endogenous regulation context of Shiga toxin retrograde trafficking (chapter 11.2). An extended discussion will be given in chapter 12.Last, a general outlook of ongoing projects is given in the perspectives section (chapter 13), in which further collaborations are highlighted.Keywords: Retrograde transport, Shiga toxin, Shiga-like toxin (SLT), STxB, syntaxin-5, Sec16A, GPP130, Retro-2, Retro-2.1, azide-functionalized Retro-2, copper-free click chemistry, small molecule target identification, mass spectrometry, non-SNARE function, anterograde trafficking inhibition, miRNA, miR199, retromer, VPS26

In photosynthetischen Organismen vermitteln Tetrapyrrole während der Chloroplastenbiogenese und als Antwort auf Veränderungen des Entwicklungszustandes sowie wechselnder Umweltbedingungen retrograde Signale zur Kontrolle der Expression von Kerngenen (NGE). In der vorliegenden Arbeit wurde ein induzierbares RNAi-System in Arabidopsis eingesetzt, um enzymatische Schritte des Mg-Zweigs der Tetrapyrrolbiosynthese (TPBS) zu inaktivieren. Es sollte gezeigt werden, inwiefern Tetrapyrrole zu einer kontinuierlichen Regulation der NGE beitragen, um abhängig von durch wechselnde Umweltbedingungen bedingte Veränderungen der TPBS, die Homöostase der Chloroplasten zu justieren. Die Untersuchungen zur Kurzzeitinduktion zeigten, dass veränderte Gehalte an Mg Protoporphyrin IX (MgP), Mg-Protoporphyrin IX-monomethylester (MgPME) und Protochlorophyllid (Pchlid) als Folge verminderter Enzymaktivitäten nicht primär zu einer Anpassung der NGE innerhalb der ersten 24 h führen. Vielmehr weisen die Ergenisse dieser Arbeit auf ein komplexes, vielschichtiges Signalnetzwerk aus retrograden und anterograden Signalwegen unter der Mitwirkung transkriptionellen und posttranslationalen Regulationsmechanismen hin. Es wird angenommen, dass Veränderungen der TPBS über ROS-Signalwege oder Redox-vermittelte Signale zur Regulation der NGE beitragen. Diese Signale könnten aus beeinträchtigten Photosystemen stammen, welche eine Folge der Akkumulation von photoreaktiven Intermediaten und unzureichenden Mengen an Chlorophyll sein können. / In photosynthetic organisms, tetrapyrroles are known to mediate retrograde control of nuclear gene expression (NGE) during chloroplast biogenesis in response to developmental and environmental changes. In these studies, an inducible RNAi system was used in Arabidopsis plants to inactivate enzymatic steps in the Mg-branch of tetrapyrrole biosynthesis (TPBS). It was intended to proof whether tetrapyrroles contribute to a permanent regulation of NGE due to changes in the TPBS and in response to environmental changes to achieve an adjustment of chloroplast homeostasis. The investigations of short-term responses due to altered levels of Mg protoporphyrin (MgP), Mg protoporphyrin monomethylester (MgPME) and protochlorophyllide (Pchlid) caused by a robust down-regulation of enzyme activity within the first 24 h reveal that these Mg porphyrins do not primarily contribute to the modulation of NGE. All results together indicate a complex multi-layered signaling network of anterograde and retrograde control and contributions of transcriptional as well as posttranslational regulation mechanisms. It is proposed that changes in the TPBS mediate the regulation of NGE via ROS-signaling pathways or redox signals deriving from disturbed photosystems due to accumulation of photoreactive intermediates and the lack of chlorophyll.

Gegenstand dieser Arbeit ist die retrograde Bewegung des Vakuumbogens, einer Bewegung des Bogenfußes entgegen der Lorentz-Kraft. Die retrograde Bewegung wird auf die Bewegung der Brennflecke des Kathodenansatzes zurückgeführt. Es wurden die Unterstrukturen der Brennflecke und ihre Bewegung im Magnetfeld untersucht. Dabei wurden bekannte makroskopische Erscheinungen (Plasmakante entlang der Magnetfeldlinien) erstmalig beobachteten mikroskopischen Erscheinungen (Jets) zugeordnet. In der vorliegenden Arbeit wurden die Eigenschaften der Jets bestimmt. Desweiteren wurde aus Untersuchungen zur Schrittweite und zeitlichen Abfolge der Brennfleckbewegung unter dem Einfluß des externen Magnetfeldes abgeleitet, daß die zufällige Bewegung des Brennflecks und seiner Bestandteile im magnetfeldfreien Fall auf kleineren Orts- und Zeitskalen als die retrograde Bewegung des Brennflecks (200...300µm) und (1...4µs) erfolgt und damit auch verschiedene Mechanismen ihre Ursache sind. Die beobachteten Jets, die aus dem Brennfleckkern emittiert werden, liefern eine Erklärung für die retrograde Bewegung. In den Jets werden elektrische Felder generiert, welche zur Neuzündung von Brennflecken beitragen. Sie sind der Hauptgrund für die Asymmetrie auf der retrograden Seite (Jets) und der Vorwärtsrichtung (keine Jets). Außerdem konnte gezeigt werden, daß bei heißen Kathoden keine Bewegungsumkehr der retrograden Bewegung erfolgt. Dadurch konnte Klarheit in Bezug auf ältere Veröffentlichungen geschaffen werden, die der retrograden Bewegung eine solche Erscheinung zugeordnet haben. / This thesis treats the phenomenon of retrograde motion of vacuum arcs, the motion of the arc root in the opposite direction of the Lorentz-force. The retrograde motion is attributed to the motion of the cathode spots. The sub-structures of cathodes spots and their motion in a magnetic field were examined. Known macroscopic phenomena (plasma edge along the magnet field lines) were related to observed microscopic phenomena (jets). These jets have been observed for the first time and their properties have been determined. From investigations of typical step widths and time constants of the cathode spots' motion in an external magnetic field it was derived, that random walk and retrograde motion of cathode spots and its fragments in the magnetic field-free case occur on smaller spatial (200...300µm) and temporal (1...4µs) scales than the retrograde motion of the spot. This leads to the conclusion that the mechanisms of both types of motion are different. The observed jets, which are emitted from the spot core, supply an explanation for the retrograde motion. Within the jets electrical fields are generated, which contribute to the ignition of new cathode spots. The jets are the reason for asymmetry on the retrograde side (jets) and the forward direction (no jets). Additionally it could be shown that with hot cathodes no reversal of the retrograde motion occurs. This cleared the assertion of older publications that a reversal of the retrograde motion is linked to the cathode temperature.

No description available.