Dynamic regulation of the actin cytoskeleton is required for synapses to form and maintain their shape. The actin cytoskeleton is regulated by Rho GTPases in response to genetic and extracellular signals. Rho GTPases are regulated by guanine nucleotide exchange factors and GTPase activating proteins (GAPs). Syd-1 is a protein that has been identified as necessary for synapse formation in worms, with similar proteins in flies, and mice. Little is known about the molecular mechanism by which Syd-1 is acting. While genetic techniques are great tools for examining synapse development, they are limited by their inability to consider the molecular nature of the protein product. By studying the biochemical nature of synaptic proteins, we can begin to understand their function with a new level of clarity. Syd-1 has a predicted Rho GAP domain; however it is thought to be inactive. The activity of the fly protein, Dsyd-1, has never been examined although it has been speculated that it is inactive in all invertebrates. Recently the mouse version was reported to have Rho GAP activity. By performing GTPase activity assays on purified proteins, I found the GAP domain of Dsyd-1 increased the GTPase activity of Rac-1 and Cdc42 but not RhoA. Members of our lab found the activity of Dsyd-1 is necessary for proper synapse formation both at the Drosophila neuromuscular junction as well as in R7 neurons. In Caenorhabditis elegans, Syd-1 was found to interact with presynaptic protein RSY-1. Since RSY-1 is evolutionarily conserved, I tested whether or not RSY-1 has a similar effect on R neurons in Drosophila. I also isolated mRNA from R neurons and evaluated the possibility of analyzing mutant neurons using comparative transcriptomics. This dissertation includes previously unpublished coauthored material.

The crayfish, Procambarus clarkii, has a multitude of ideal sites in which synaptic transmission may be studied. Its opener muscle, being innervated by a single excitatory neuron is a good model for studying the structure/function of neuromuscular junctions since the preparation is identifiable from animal to animal and the nerve terminals are visible using a vital dye. This allows ease in finding a suitable site to record from in each preparation and offers the ability to relocate it anatomically. Marking a recorded site and rebuilding it through electron microscopy gives good detail of synaptic struture for assesment.In the first of these studies, low output sites known as stems (which lie between varicosities) were used to reduce n (number of release sites) in order to minimize synaptic complexity so individual quantal events could be analyzed by their unique parameters (area, peak, tau, rise time and latency). This was in attempt to uncover specific quantal signatures that could be traced back to the structure of the area recorded. It was found that even at stem regions synaptic structure is still complex having multiple synapses each of which could harbor a number of AZs. This gives insight as to how quantal analysis should be treated. Even low output synapses n must be treated at the AZ level.Synaptic depression was studied at the crayfish extensor muscle. By depressing the phasic neuron and recording from the muscle it appears thatdepression is a presynaptic phenomenon. The use of 5-HT gave insight to vesicular dynamics within the nerve terminal, by delaying depression and increasing maximum EPSP amplitude. TEM of phasic nerve terminals reveals no change in numbers of dock or RRP vesicles. Short term facilitation and vesicular dynamics were studied with the use of 5-HT and a neurotoxin TBOA, which blocks the glutamate transporter. In this study I showed differential mechanisms that control RRP and RP vesicles. By blocking glutamate reuptake, the RRP is depleted as shown by reduced EPSPs, but recovered with 5-HT application. The understanding of vesicle dynamics in any system has relevance for all chemical synapses.

Synapse development is a stepwise process that requires the recruitment of key synaptic components to active zones, followed by continual maintenance of these structures to maintain connectivity and stability throughout the life of the organisms. Early synapse development requires the recruitment of early scaffolding proteins to establish stable connectivity as well as provide sites of recruitment of other vital synaptic proteins. One of the earliest proteins to be localized to the synapse is the conserved protein Syd-1. Syd-1 proteins contain a Rho GTPase activating protein (GAP)-like domain of unclear significance. Here I show that Drosophila Syd-1 interacts with all six fly Rhos and has GAP activity towards RAC1. I then show that lacks GAP activity localizes normally to presynaptic sites and is sufficient to recruit Nrx-1 but fails to cluster Brp normally and genetically interacts with RAC1 in vivo. I conclude that contrary to previous models, the GAP domain of fly Syd-1 is active and required for presynaptic development. Additionally, I’ve identified a previously uncharacterized protein, Vezl, as being critical for retrograde axonal transport and synaptic maintenance. I found that Vezl required for normal neuronal growth and that vezl loss resulted in decreased neuron size and the formation of swollen neuronal terminals that accumulated membrane markers and axonal transport cargo. I found that vezl mutants specifically retrograde transport of cargo and particularly affected signaling endosomes. The signaling endosomes were unable to initiate retrograde transport in vezl mutants and remained stuck within the distal boutons unable to relay their signaling peptides back to the nucleus. I conclude that Vezl is serving a role in attaching retrograde cargo to dynein and the microtubules specifically at neuron tips so that they can undergo retrograde axonal transport. This dissertation includes previously published and unpublished co-authored material. / 2020-09-06

Sismotectonique, paléotsunami et le tsunami scénarios sont examinés sur la côte du Nord de l'Égypte dans le cadre du tsunami européen ASTARTE projet et le projet IMHOTEP français-égyptiens. La géologie, la géomorphologie, séismicité, des mécanismes focaux, l'inversion de stress calculée et des données GPS utilisée pour identifier le régime de stress de jour présent des zones actives et les zones de tsunamigène. Tranchées et carottes ont été creusées à deux sites. Le balayage de radiographie, la sensibilité magnétique, l'analyse de taille de grain, l'échantillonnage, macrofossile détections, total des matériaux organiques et inorganiques et la datation au carbone est effectuée pour identifier les signatures tsunami. La couche sablonneuse blanche de haute énergie riche en fossiles retravaillés est corrélée avec le 21 juillet 365 dans le Kefr Saber. Les quatre couches sédimentaires de haute énergie à l'El Alamein sont corrélées les tsunamis historiques de 1600 avant J.C., le 21 juillet 365, 8 août 1303, le 24 juin 1870. / Seismotectonic, paleotsunami deposits and tsunami scenarios are investigated along the north coast of Egypt in the framework of the tsunami ASTARTE European and the French-Egyptian IMHOTEP projects. The geology, geomorphology, seismicity, focal mechanisms, calculated stress inversion, and GPS data were used to identify the present day stress regime of the main active zones and the tsunamigenic zones. Trenches and cores were dug in Kefr Saber and EL Alamein sites. X-ray scanning, magnetic susceptibility, grain size analysis, sampling, macrofossil detections, XRD analysis, total organic and inorganic matter measurements and carbon dating are carried out to identify the paleotsunami signatures. The high-energy white sandy layer rich in reworked fossils at Kefr Saber are correlated with 21 July 365, while the four characteristic high-energy sedimentary layers at the El Alamein site are correlated with the historical tsunami events of 1600 BC, 21 July 365, 8 August 1303, and 24 June 1870.