Epileptogenesis is the complex process of the brain developing epileptic acitivity. Due to the role of glutamate and the hippocampus in synaptic plasticity a dysregulation in glutamate neurotransmission and hippocampal dysfunction are implicated in the process of epileptogenesis. However, the exact causal factors that promote epileptogenesis are unknown. We study presynaptic proteins that regulate glutamate neurotransmission and their role in epileptogenesis. The presynaptic protein, tomosyn, is believed to be a negative regulator of glutamate neurotransmission; however, no one has studied the effects of this protein on glutamate transmission in vivo. Furthermore, evidence suggests that mice lacking tomosyn have a kindling phenotype. Thus, in vivo glutamate recordings in mice lacking tomosyn have the potential to elucidate the exact role of tomosyn in glutamate neurotransmission and its potential relationship to epileptogenesis. Here we used biosensors to measure glutamate in the dentate gyrus (DG), CA3, and CA1 of the hippocampus in tomosyn wild-type (Tom+/+), heterozygous (Tom+/-), and knock out (Tom-/-) mice. We found that, in the DG, that glutamate release increases as tomosyn expression decreases across genotype. This suggests that tomosyn dysregulation in the DG leads to an increase in glutamate release, which may explain why these mice have an epileptogenic phenotype.

Tomosyn is a protein belonging to the Lgl family and conserved across the animal and plant kingdom. Tomosyn is composed of N-terminal domain containing WD40 motif and Cterminal domain, where the R-SNARE motif is located at the end of the C-terminal domain. This motif is classified as homologous to R-SNARE motif of synaptobrevine, which is a protein located on the surface of the vesicles and participating in the formation of SNARE complex and subsequent fusion of the vesicles with the plasma membrane. Thus the role of this tomosyn is mainly the regulation of exocytosis. Apart from the animal tomosyn, its yeast homolog Sro7/Sro77 is also examined, however during the evolution it has lost its R-SNARE motif and therefore the plant tomosyns remain unexplored. The aim of this diploma thesis was to characterize both of the plant tomosyns: AtTYN1 and AtTYN2 in Arabidopsis thaliana plant. The experimental thesis included the bioinformatic analysis, the DNA construct creation, a search for interactors by yeast two-hybrid system and monitoring the localization using the confocal microscope. The bioinformatic analysis results, including the creation of phylogenetic tree, not only revealed the conservation of tomosyns across the different classes, but also the division of both tomosyns into different clusters. There was...

No description available.

Platelet secretion is important for hemostasis and thrombosis. The components released are also involved in atherosclerosis, inflammation, angiogenesis, and tumor growth. Though the exact mechanism(s) of platelet secretion is still elusive, accumulating evidence demonstrates that SNAREs (Soluble N-ethylmaleimide Sensitive Factor Associated Receptor) and their regulatory partners are critical for platelet exocytosis. Formation of a trans-bilayer complex composed of one v-SNARE (i.e. VAMPs) and two t-SNAREs (i.e. syntaxin and SNAP-25-type) is minimally required for membrane fusion. Regulatory proteins control the rate and specificity of the complex assembly. VAMP-8 and SNAP-23 (a SNAP-25-type t-SNARE) are clearly important; however, the identity of the functional syntaxin has been controversial. Previous studies, using anti-syntaxin antibodies in permeabilized platelets, suggested roles for both syntaxin-2 and -4. These conclusions were experimentally tested using platelets from syntaxin knockout mice and from a Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL4) patient that lacks syntaxin-11. Platelets from syntaxin-2 and syntaxin-4 single or double knockout mice had no significant secretion defect. However, platelets from the FHL4 patient had a robust defect, though their morphology, activation, and cargo levels appeared normal. Semi-quantitative western blotting showed that syntaxin-11 is the most abundant syntaxin in both human and murine platelets. Co-immunoprecipitation experiments showed that syntaxin-11 forms SNARE complexes with VAMP-8 and SNAP-23. These data conclusively demonstrate that syntaxin-11, but not syntaxin-2, or -4, is required for platelet exocytosis. We also show that a syntaxin binding protein, tomosyn-1, is important for platelet exocytosis and hemostasis. Tomosyn-1 was identified from platelet extracts using affinity chromatography, RT-PCR analysis, and western blotting analysis. Tomosyn-1 was co-immunoprecipitated with syntaxin-11/SNAP-23 from both resting and activated platelet extracts. Platelets from tomosyn-1-/- mice displayed a secretion defect, but their morphology and activation appeared normal. Tomosyn-1-/- mice showed impaired thrombus formation in two different injury models. Given the importance of platelet secretion to hemostasis, it is hoped that the insights gained from these studies in this dissertation will help to identify new and more valuable therapeutic targets to control clot formation.