Refine
Document Type
- Doctoral Thesis (3)
Language
- English (3)
Has Fulltext
- yes (3)
Is part of the Bibliography
- no (3)
Institute
An essential part of the animal survival strategy comprises the ability to control body movement and coordinate long-term navigational strategies, in order to maintain locomotion towards a nutrition source and stay in its vicinity. In the nematode Caenorhabditis elegans (C. elegans) this function is carried out by neuronal circuits, that vary their activity in response to diverse environmental condition.
This comprises different classes of neurons, acting together in a sensory, signaling and modulatory system to control body posture and induce behavioral responses. For this reason, one particular goal in the field of neuroscience research is to elucidate the mechanisms of how neuronal circuits integrate multiple sensory cues to navigate the environment. Aim of this study was to analyze the function of a neuronal network comprising the interneurons AVK, as well as the identification of signaling molecules, controlling body posture during food related locomotory behavior. This should be achieved by establishing optogenetic approaches, which provide a non inversive and temporally precise control of neuronal activity and drives the activation or silencing of individual neurons, to alter the neuronal basis of behavior. Animals exposed to food perform a dwelling-like behavior, characterized by a slowing of locomotion with a reduced crawling distance and an irregular movement, accompanied by a high frequency of pauses, reversals and directional changes. Upon food-removal, they initiate a local-search behavior with the same behavioral characteristics, but with a more pronounced sinusoidal movement. After a prolonged period of unsuccessful food finding, animals exhibited long runs with reduced pauses, reversals and turnings, increasing their maximal covered distance, indicated as dispersal behavior. Acute photoinhibition of AVK neurons, mediated by cell-specific expression of halorhodopsin (NpHR) caused the animals to perform a dwelling-like locomotory state with increased bending angles, as seen during local-search behavior. Thus, food-induced behavioral effects are mimicked by the optogenetic manipulation of AVK interneurons.
In this study, signaling molecules were ascertained by cell specific mRNA profiling of AVK neurons, mediating these behavioral responses. It was able to demonstrate, that flp-1, coding for a FMRFamidelike neuropeptide, is one of the genes with the highest distribution in AVK. In the absence of food, AVK neurons continuously release the FMRFamide-like neuropeptide FLP-1 to inhibit a subset of target motoneurons, leading the animals to maintain a low body curvature to promote dispersing behavior.
Conversely, if AVK was inhibited by NpHR or the presence of food, less FLP-1 was secreted to the body fluid, indicated by reduced intracellular fluorescence levels of mCherry-tagged FLP-1 proteins in the scavenger cells. The search of a FLP-1 receptor was successful by in vitro investigation on G protein-coupled receptors (GPCRs) and neuropeptide ligands, revealing NPR-6 to be activated by FLP-1 neuropeptides, but with a low potency. Expression pattern of the NPR-6 receptor indicated receptor localization in in the VC ventral cord and SMB head motoneurons, as well as in a subset of other neurons required for chemosensation and feeding. AVK interneurons are highly coupled to SMB head motoneurons, forming electrical synapses composed of the gap junction protein subunits UNC-7 and UNC-9. Elimination of SMB or gap junction genes using cell ablation and RNA interference, respectively, phenocopied effects of AVK inhibition on bending angles. Furthermore, this study was able to demonstrate that these neurons get inhibited during FLP-1 transmission to the NPR-6 receptor, which was required to mediate AVK effects on crawling behavior. Consequently, photoinhibition of AVK caused disinhibition of VC and SMB neurons, in order to enhance sinusoidal movement and to induce a local-search related locomotory behavior.
Thereby, FLP-1 neuropeptide transmission is the preferred used signaling pathway over direct gap junction coupling. Additional neuropeptides and receptors were identified to be essential downstream to AVK neurons to mediate effects on body curvature and locomotory behavior as well. The high-potency FRPR-7 receptor was shown to mediate FLP-1 peptide effects on undulatory motion during swimming in a liquid environment, rather than crawling locomotion on a solid surface. This result suggests that the receptor NPR-6 is required for FLP-1 peptide effects on bending and crawling locomotion, whereas conversely the receptor FRPR-7 is addressed by FLP-1 peptides to exclusively regulate swimming behavior. The FRPR-7 receptor is expressed in the AIM and NSM motoneurons, which are suggested to be the primary neuronal candidates mediating swimming behavior. Furthermore, this study provides evidence, that FRPR-7 acts in the DVC interneuron to control spontaneous reversal behavior, most probably by inhibitory FLP-1 signaling from the AVK neurons. Among other neuropeptides, the FMRFamide-like peptide FLP-26 binds with higher affinity to NPR-6 receptors than FLP-1 peptides. FLP-26 peptides are expressed in the SMB motoneurons, where they are able to further potentiate FLP-1 inhibitory effects by simultaneous binding to NPR-6.
...
In der vorliegenden Arbeit wurde ein integrativer Netzwerkmodellierungsansatz gewählt, um die Rolle des Endothels im Kontext der Arteriosklerose zu untersuchen. Hierbei wurden bioinformatische Analysen, laborexperimentelle Versuche und klinische Daten vereinigt und aus dieser Synthese neue klinisch relevante Gene identifiziert und beschrieben.
Das Endothel trägt maßgeblich zur Homöostase des vaskulären Systems bei und eine Dysfunktion des Endothels fördert die Entstehung der Arteriosklerose. Im Zuge der Atherogenese entstehen vermehrt reaktive Sauerstoffspezies, die Lipide in der Membran von Plasma-Lipoprotein-Partikeln und in der zellulären Plasmamembran oxidieren. Eine Gruppe solcher oxidierter Membranlipide ist oxPAPC, das in erhöhter Konzentration in arteriosklerotischen Plaques und lokal an Orten chronischer Entzündung im vaskulären System vorkommt. Weitherhin findet sich diese Gruppe von oxidierten Phospholipiden in oxidierten LDL-Partikeln, in denen oxPAPC die Bindung an Makrophagen vermittelt und hierdurch maßgeblich zur Bildung der Schaumzellen und damit zum arteriosklerotischen Prozess beiträgt. Die durch oxPAPC verursachte Veränderung der Endothelzelle ist bisher wenig erforscht. Es ist jedoch bekannt, dass oxPAPC die Transkriptionslandschaft in Endothelzellen tiefgreifend verändert. Um der Komplexität der Endothelzellveränderung gerecht zu werden, wurde ein bayesscher Ansatz angewendet.
In einem ersten Schritt wurden Expressionsprofile von humanen Aortenendothelzellen (HAEC) aus 147 Herztransplantatspendern verwendet. Diese Expressionprofile enthalten Transkriptionsinformationen der 147 HAEC, die mit oxPAPC oder Kontrollmedium behandelt worden waren. Es wurden signifikant koexprimierte Gene identifiziert und hiervon Gen-Paare berechnet, die einen differentiellen Vernetzungsgrad zwischen Kontroll- and oxPAPC-Status aufweisen. Dieses Netzwerkmodell gibt darüber Aufschluss, welche Gene miteinander in Verbindung stehen. 26759 Gene-Paare, die differentiell verbunden und signifkant koexprimiert waren, wurden hierarchisch gruppiert. Es wurden neun Gen-Gruppen mit einer erhöhten und elf Gen-Gruppen mit einer verminderten Konnektivität nach oxPAPC identifiziert. Gruppe 6 der erhöhten Konnektvitäts-Gruppen wies hierbei die höchste kohärente Konnektivität von allen Gruppen auf. Eine Analyse signifikant überrepräsentierter kanonischer Gensätze ergab, dass diese Gruppe insbesondere Serin-Glycin-Aminosäuremetabolismus, tRNA- und mTOR-Aktivierung wiederspiegelte. Der hier gewählte Netzwerkmodellierungsansatz zeigte auf, dass der Aminosäuremetabolismus durch oxidizerte Phospholipide massiven Veränderungen unterworfen ist.
Um den Mechanismus der Veränderung des Aminosäuremetabolismus näher zu untersuchen, wurden bayessche Netzwerkmodelle verwendet. Dieses Netzwerkmodell enthält im Gegensatz zum differentiellen Koexpresssionsmodell gerichtete Informationen innerhalb des Netzwerkgraphes. Die Gen-Gen Verbindungen sind kausal, wodurch sich eine Hierarchie bildet und Schlüsselfaktoren innerhalb des Netzwerks bestimmt werden können. Durch die Integrierung von Expressionsprofilen und Genomprofilen derselben HAEC-Kohorte und der Inferenz von kausalen Gen-Gen-Verbindungen ergaben sich zwei bayessche Netze: Kontroll- und oxPAPC-Netzwerk. Permutationsuntersuchungen und systematische Beurteilung im Vergleich zu Gen-Gen-Verbindungen in Online-Datenbanken zeigten eine erhöhte Prognosefähigkeit der beiden HAEC bayesschen Netze. Es wurden die Schlüsselfaktoren und deren Teilnetzwerke berechnet und auf biologische Wege hin untersucht. Hierbei wurde das mitochondriale Protein MTHFD2 als ein Schlüsselfaktor für ein Teilnetzwerk des oxPAPC bayesschen Netzes identifiziert. Dieses Teilnetz zeigte eine ähnliche Gensatzanreicherung wie GOC-AA und überlappte mit diesem signifikant.
...
Structure-function relationships in substrate binding protein dependent secondary transporters
(2023)
This work provides new insights into the relevance of SBP dependent secondary transport systems, especially in the thus far under-researched subgroup of TAXI transporters. Importantly, we identified and characterized the TAXI transport system TAXIPm-PQM from Proteus mirabilis. We demonstrated that, in contrast to previously characterized SBP dependent secondary transport systems, TAXIPm-PQM is a proton coupled system and transports the C5-dicarboxylate α- ketoglutarate. Since initially the transport of α-ketoglutarate could only be demonstrated in vivo but not in vitro using established protocols (Mulligan et al. 2009), we investigated in detail the differences between the in vivo and in vitro assay. This resulted in a bioinformatic analysis of TRAP and TAXI signal peptides, which strongly implied that TAXIPm-P requires a transmembrane anchor to allow for transport. We then provided TAXIPm-P surface tethered to the membrane in in vitro transport assays and confirmed the prediction of our bioinformatic analysis that TAXIPm-PQM deploys a membrane-anchored instead of a soluble SBP. Furthermore, the TAXI transport system TAXIMh-PQM from Marinobacter hydrocarbonoclasticus transports fumarate only if both membrane domains Q and M are present. For further characterization, Michaelis-Menten kinetics and affinities were determined for both TAXI transport systems TAXIPm-PQM from Proteus mirabilis and TAXIMh-PQM from Marinobacter hydrocarbonoclasticus. In addition, nanobodies were selected for the membrane domain TAXIPm-QM from Proteus mirabilis to stabilize different conformations which can serve in subsequent structural elucidation studies. Furthermore, the TRAP SBP TRAPHi-SiaP from Haemophilus influenzae was shown to interact not only with its corresponding membrane domain TRAPHi-SiaQM but with at least one additional transporter. It was thereby excluded that TRAPHi- SiaP transfers N-acetylneuraminic acid to the only native E. coli TRAP transporter TRAPEc-YiaMNO and suggested to rather interact with a SBP dependent ABC transport system as this protein family represents the largest SBP dependent protein group in E. coli (Moussatova et al. 2008).