A Novel Communication Mechanism Between the Presynapse and Postsynapse Through Exosomes: A Dissertation

The minimal element of the nervous system, the synapse, is a plastic structure that has the ability to change in response to various internal and external factors. This property of the synapse underlies complex behaviors such as learning and memory. However, the exact molecular and cellular mechanisms involved in this process are not fully understood. To understand the mechanisms that regulate synapse development and plasticity I took advantage of a powerful model system, the Drosophila larval neuromuscular junction (NMJ). In this system, both anterograde and retrograde signaling pathways critical for coordinated synapse development and plasticity have been documented. An anterograde WNT/Wingless (Wg) signaling pathway plays a crucial role in both developmental and activity-dependent synaptic plasticity at the NMJ. Presynaptic motor neuron terminals secrete highly hydrophobic Wg, which travels to relatively distant postsynaptic sites where it activates a signal transduction pathway required for postsynaptic development. In the first half of my thesis I unraveled a previously unrecognized cellular mechanism by which Wg is shuttled to postsynaptic sites. In this mechanism Wg rides on secreted microvesicles or exosomes that contain a dedicated WNT secretion factor, the WNT-binding transmembrane protein, Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). To our knowledge, this was the first in vivo study demonstrating that neurons release exosomes, which are involved in trans-synaptic communication. Moreover, this was the first study showing that hydrophobic WNT signals are transported to the extracellular space on exosomes to reach WNT-receptor containing target cells. Retrograde signals are also critical during development and plasticity of synaptic connections. These signals function to adjust the activity of presynaptic cells according to postsynaptic cell outputs, to maintain synaptic function within a dynamic range. However, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are not clear. In the second half of my thesis, I provided evidence that a crucial component of retrograde signaling at the fly NMJ, Synaptotagmin-4 (Syt4), is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Drosophila Syt4 is known to reside on postsynaptic vesicles at the NMJ and function as a calcium sensor to release a retrograde signal upon synaptic activity. This event is required for coordinated maturation of the presynaptic terminal. We demonstrated that retrograde Syt4 function in postsynaptic muscle is required for activity-dependent presynaptic growth. However, surprisingly, Syt4 protein was not synthesized in postsynaptic muscles. Instead, Syt4 was produced in motorneurons and transferred to postsynaptic muscle cells via exosome secretion by presynaptic cells. The above study provided evidence for a presynaptic control of postsynaptic retrograde signaling through exosomal transfer of an essential retrograde signaling component. In summary, this body of work reveals a novel mechanism of trans-synaptic communication through exosomes. While intercellular communication through exosomes had been demonstrated during antigen presentation in the immune system, our studies were the first to substantiate this mode of communication in the nervous system. Thus, these studies provide a significantly deeper and novel understanding of the mechanisms underlying synapse development and plasticity

Long alkyl group containing benzoyl-barbituric acid derivative and synthesis there of borane complex

Barbituric acids have long been known for many years and it has several biological activity. Barbituric acids are used in many areas of medicine and pharmaceutical industry. Surprisingly, there are several study about barbituric acid derivatives in literatures, only few examples deal with liquid crystal properties. In this study, long alkyl group containing benzoyl-barbituric acid derivatives and their boron complexes are synthesized. Structural characterizations of new compounds were made with 1H, 13C and 11B NMR, FT-IR and MS spectroscopy. Moreover, liquid crystal properties were examined by polarized optical microscope.Barbituric acids have long been known for many years and it has several biological activity. Barbituric acids are used in many areas of medicine and pharmaceutical industry. Surprisingly, there are several study about barbituric acid derivatives in literatures, only few examples deal with liquid crystal properties. In this study, long alkyl group containing benzoyl-barbituric acid derivatives and their boron complexes are synthesized. Structural characterizations of new compounds were made with 1H, 13C and 11B NMR, FT-IR and MS spectroscopy. Moreover, liquid crystal properties were examined by polarized optical microscope

WNTs tune up the neuromuscular junction

Although WNTs have been long thought of as regulators of cell fate, recent studies highlight their involvement in crucial aspects of synaptic development in the nervous system. Particularly compelling are recent studies of the neuromuscular junction in nematodes, insects, fish and mammals. These studies place WNTs as major determinants of synapse differentiation and neurotransmitter receptor clustering

Pulsed Infrared Laser Crystallization Mechanism of Amorphous Silicon Thin-Films

Laser crystallization of amorphous silicon layers is an active research field in photonic applications. In literature, typically single laser pulses are used to examine the absorption mediated crystallization of amorphous silicon. Here, the slow quenching regime dynamics for laser crystallization with highly overlapping infrared nanosecond pulses was examined experimentally and theoretically. In contrast to commonly used >10 micrometer thick layers oñ 700°C heated substrates, in our study, the thicknesses of deposited amorphous Si layers were decreased down to 1 micrometer range, and the laser crystallization was performed at room temperature. Increasing the use of Si in thin-film transistors and photovoltaic applications drive researchers to find cost-effective and efficient ways of manufacturing crystalline Si films on various types of substrates. Understanding the mechanism of the laser crystallization process of Si films by pulsed lasers becomes crucial. This work reveals the laser crystallization mechanism of Si thin films in macroscopic scales by considering heat transfer and accumulation dynamics. Our motivation is to describe the dynamics of the laser crystallization of Si films to provide a complimentary guide to produce device-grade crystal Si films by infrared pulsed laser without employing preheated substrates

Fethi Gürcan biyografisi : bir ihtilalcinin hayatı

Ankara : İhsan Doğramacı Bilkent Üniversitesi İktisadi, İdari ve Sosyal Bilimler Fakültesi, Tarih Bölümü, 2014.This work is a student project of the The Department of History, Faculty of Economics, Administrative and Social Sciences, İhsan Doğramacı Bilkent University.by Ünsal, Mehmet Süha

Laser Crystallization of Amorphous Ge Thin Films via a Nanosecond Pulsed Infrared Laser

Understanding the dynamics of the laser crystallization (LC) process of Ge thin films by nanosecond (ns) pulsed infrared (IR) lasers is important for producing homogeneous, crack-free crystalline device-grade films for use in thin-film transistors, photo-detectors, particle detectors, and photovoltaic applications. Our motivation is to describe a ns IR laser-based crystallization process of Ge by implementing suitable parameters to fabricate thin-film devices. Our LC technique was applied to crystallize thin amorphous Ge (a-Ge) films with thicknesses suitable for device applications. The LC process was applied to a 300 nm-thick a-Ge thin film utilizing a 200 ns pulsed IR laser with a wavelength of 1064 nm. Electron-beam-evaporation-deposited a-Ge on glass substrates were subject to successive ns laser pulses with a line focus. The crystallinity of the polycrystalline Ge (pc-Ge) films was evaluated by Raman spectroscopy, optical microscopy, and electron backscatter diffraction (EBSD). LC-Ge exhibited a Raman peak of around 300 cm(-1), confirming successful crystallization of a-Ge. pc-Ge domain sizes exceeding several tens of micrometers were observed in EBSD scans. LC of a-Ge minimizes the thermal energy budget of processing and provides flexibility to locally crystallize the film. Our work is the first demonstration of the LC of a-Ge thin films, resulting in domain sizes exceeding tens of micrometers via a ns pulsed IR laser

Regulation of postsynaptic retrograde signaling by presynaptic exosome release

Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling

Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless

Wnts play pivotal roles during development and in the mature nervous system. However, the mechanism by which Wnts traffic between cells has remained elusive. Here we demonstrate a mechanism of Wnt transmission through release of exosome-like vesicles containing the Wnt-binding protein Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). We show that at the Drosophila larval neuromuscular junction (NMJ), presynaptic vesicular release of Evi is required for the secretion of the Wnt, Wingless (Wg). We also show that Evi acts cell-autonomously in the postsynaptic Wnt-receiving cell to target dGRIP, a Wg-receptor-interacting protein, to postsynaptic sites. Upon Evi loss of function, dGRIP is not properly targeted to synaptic sites, interfering with postsynaptic Wnt signal transduction. These findings uncover a previously unknown cellular mechanism by which a secreted Wnt is transported across synapses by Evi-containing vesicles and reveal trafficking functions of Evi in both the Wnt-producing and the Wnt-receiving cells. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online

Mechanism of evenness interrupted (Evi)-exosome release at synaptic boutons

Wnt signaling plays critical roles during synaptic development and plasticity. However, the mechanisms by which Wnts are released and travel to target cells are unresolved. During synaptic development, the secretion of Drosophila Wnt1, Wingless, requires the function of Evenness Interrupted (Evi)/Wls, a Wingless-binding protein that is secreted along with Wingless at the neuromuscular junction. Given that Evi is a transmembrane protein, these studies suggested the presence of a novel vesicular mechanism of trans-synaptic communication, potentially in the form of exosomes. To establish the mechanisms for the release of Evi vesicles, we used a dsRNA assay in cultured cells to screen for genes that when down-regulated prevent the release of Evi vesicles. We identified two proteins, Rab11 and Syntaxin 1A (Syx1A), that were required for Evi vesicle release. To determine whether the same mechanisms were used in vivo at the neuromuscular junction, we altered the activity of Rab11 and Syx1A in motoneurons and determined the impact on Evi release. We found that Syx1A, Rab11, and its effector Myosin5 were required for proper Evi vesicle release. Furthermore, ultrastructural analysis of synaptic boutons demonstrated the presence of multivesicular bodies, organelles involved in the production and release of exosomes, and these multivesicular bodies contained Evi. We also used mass spectrometry, electron microscopy, and biochemical techniques to characterize the exosome fraction from cultured cells. Our studies revealed that secreted Evi vesicles show remarkable conservation with exosomes in other systems. In summary, our observations unravel some of the in vivo mechanisms required for Evi vesicle release