Post-translational protein modifications (PTM) vastly increase the complexity and functional diversity of the proteome, to precisely regulate crucial cellular processes. The plant immune system is composed of complex signalling networks that are influenced by various PTMs. Activation of plant immunity is associated with a rapid burst of nitric oxide (NO), which can covalently modify cysteine thiols within target proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs), constituting a redox-based PTM. Another key PTM involved in plant immunity is SUMOylation, an essential mechanism involving the conjugation of the small ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins. Although the targets and mechanisms of S-nitrosylation and SUMOylation are becoming evident, how these key PTMs are themselves regulated remains obscure. Work presented in this thesis reveals that during plant immune signalling, the sole Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO conjugating activity in vitro and mutational analysis revealed that the site of this modification, Cys139, is not required for enzyme activity but rather constitutes a redox-sensitive inhibitory switch. Generation and characterization of transgenic Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1 revealed that Cys139 is required for efficient immunity against bacterial pathogens. Furthermore, after immune activation, S-nitrosylation of this residue inhibits global SUMOylation of proteins. These results provide evidence of a novel means of crosstalk between S-nitrosylation and SUMOylation in the context of plant immunity. The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione (GSNO), which is thought to constitute a stable reservoir of NO bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which indirectly influences the levels of protein SNOs. In this study, transgenic plants overexpressing FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds, including nitric oxide overproducer 1 (nox1), to further investigate the roles of GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression completely recovers developmental and disease susceptibility phenotypes of gsnor1, but not nox1 mutant plants, highlighting in vivo differences between accumulation of GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation of GSNOR1, inhibiting its enzymatic activity. This suggests a previously unreported means by which NO might regulate its own bioavailability. Further work in this study revealed that recombinant GSNOR1 can be SUMOylated in vitro, which appeared to increase its enzymatic activity. Several potential SUMO modification sites were identified within GSNOR1 and mutational analysis revealed that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo, although the site(s) and biological function of SUMOylation were not identified. Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation and SUMOylation.

Rapid production of type I Interferon is pivotal to initiate cellular antiviral host defense and adaptive immunity. In order to facilitate innate immune processes, a cell harbors pattern recognition receptors (PRRs) which sense distinctive forms of pathogen associated molecular patterns (PAMPs). For example, Toll like receptors (TLRs) and RIG-I like receptors (RLRs) were discovered as PRRs for pathogen derived molecules and the production of type I Interferon (IFN). To induce type I IFN, several transcription factors such as nuclear factor-kappaB (NF-ĸB), interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), and activating protein-1 (AP-1) need to be stimulated through the specific signaling adaptors. Among them, our lab is interesting in the death domain (DD) containing proteins Receptor interacting kinase1 (RIP1) and Fas-associated death domain protein (FADD), which we showed were important for innate signaling processes. RIP1 and FADD were initially identified as Fas and TNFR interacting proteins which were involved in death receptor mediated apoptosis. Aside from apopotic function, recent publications indicate that RIP1 and FADD mediate cell survival, proliferation, and cytokine production through NF-ĸB activation. Here, we show that RIP1 and FADD are essential for efficient TLR-independent signaling. We report that RIP1 and FADD lacking MEF cells are sensitive to viral cytolysis and also exhibit impaired IFN production against dsRNA virus infection. RIP1 acts as a scaffolding protein for death receptor mediated apoptosis and NF-ĸB activation, necrosis, and innate immunity. As mentioned, we demonstrate that cells lacking RIP1 are sensitive to RNA virus infection. To understand the detailed mechanisms of RIP1 function in innate signaling, we first tested whether RIP1 is involved in RIG-I signaling. We found that RIP1 forms a complex with RIG-I in the presence of dsRNA. Additionally, we showed that RIP1 is required for optimal RIG-I and melanoma differentiation-associated protein 5 (MDA-5) activity. We also find that FADD, a RIP1 interaction protein, is implicated in innate immunity. To study the precise mechanisms of FADD in type I IFN signaling, we generated FADD variants and used luciferase reporter assays to indicate that the FADD death effector domain (DED) is crucial for IFN-β signaling. In order to identify interacting partners of FADD, yeast two hybrid assays were performed and indicated that FADD binds to protein inhibitor of activated STAT (PIAS1), part of the SUMO machinery. SUMOylation is a reversible post-translational modification of a protein by SUMO, a 100 amino acid protein. The consequence of SUMOylation alters specific proteins’ function by affecting activity, localization, stability or influencing molecular interactions by interfering with or linking to a target protein. To confirm FADD-PIAS interactions, we conducted in-vitro SUMOylation assays by using Ubc9 conjugated FADD and found possible FADD SUMOylation sites. We also discovered that FADD and SUMO are co-localized in the nucleus. This result reveals that FADD undergoes SUMOylations and its modification might regulate FADD’s function, including role in innate signaling. Furthermore, we report here that HTLV-1 Tax protein interacts with RIP1 and inhibits IFN-β inducing signaling by abrogating RIP1 and IRF7 interaction. This implies that RIP1 is involved in the regulation of IRF7 and is essential for IFN-β production. Collectively, our data demonstrate the significance of RIP1 and FADD in dsRNA recognition pathways in mammalian cells that are essential for the optimal induction of type I IFNs and other innate genes important for host defense.

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that can be covalently conjugated to a variety of “target” molecules to modulate their turnover rate, localization and/or function. The full range of Ubl functions is only beginning to be understood. The Raught lab is using mass spectrometry and high throughput screening methods, along with standard cell biology and biochemistry approaches, to better understand Ubl function. Here, I describe the role of a Ubl called small ubiquitin-related modifier (SUMO) in the budding yeast alcohol stress response. We identified a regulatory mechanism of the SUMO system, involving modulation of the localization of a SUMO protease. Secondly, using mass spectrometry (MS), I assisted in identifying several yeast and mammalian Ubl “chain” linkages. Finally, I propose an integrated MS methodology designed to complement standard database software for the confident identification of Ub/Ubl conjugation sites.

Microscopic traffic simulation is useful tool in analysing traffic and estimating the capacity and level of service of road networks. In this thesis, the four legged Idrottsparken roundabout in the city of Norrkoping in Sweden is analysed by using the microscopic traffic simulation package AIMSUN. For this purpose, data regarding traffic flow counts, travel times and queue lengths were collected for three consecutive weekdays during both the morning and afternoon peak periods. The data were then used in model building for simulation of traffic of the roundabout. The Root Mean Square Error (RMSE) method is used to get the optimal parameter value between queue length and travel time data and validation of travel time data are carried out to obtain the basic model which represents the existing condition of the system. Afterward, the results of the new models were evaluated and compared to the results of a SUMO model for the same scenario model. Based on calibrated and validated model, three alternative scenarios were simulated and analysed to improve efficiency of traffic network in the roundabout. The three scenarios includes: (1) add one free right turn in the north and east sections; (2) add one free right turn in the east and south sections; and (3) addition of one lane in roundabout. The analysis of these scenarios shows that the first and second scenario are only able to reduce the queue length and travel time in two or three legs, while the third scenario is not able to improve the performance of the roundabout. In this research, it can be concluded that the first scenario is considered as the best scenario compared to the second scenario and the third scenario. The comparison between AIMSUN and SUMO for the same scenario shows that the results have no significance differences. In calibration process, to get the optimal parameter values between the model measurements and the field measurements, both of AIMSUN and SUMO uses two significantly influencing parametersfor queue and travel time. AIMSUN package uses parameter of driver reaction time and the maximum acceleration, while SUMO package uses parameter of driver imperfection and also the driver rection time.

Ubiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that can be covalently conjugated to a variety of “target” molecules to modulate their turnover rate, localization and/or function. The full range of Ubl functions is only beginning to be understood. The Raught lab is using mass spectrometry and high throughput screening methods, along with standard cell biology and biochemistry approaches, to better understand Ubl function. Here, I describe the role of a Ubl called small ubiquitin-related modifier (SUMO) in the budding yeast alcohol stress response. We identified a regulatory mechanism of the SUMO system, involving modulation of the localization of a SUMO protease. Secondly, using mass spectrometry (MS), I assisted in identifying several yeast and mammalian Ubl “chain” linkages. Finally, I propose an integrated MS methodology designed to complement standard database software for the confident identification of Ub/Ubl conjugation sites.

SUMO is a small ubiquitin-like modifier. The fluorescent fused SUMO (active for sumoylation) localized in the nucleus, while C-terminal truncated SUMO (inactive for sumoylation) diffused in the cytoplasm. Daxx is a SUMO target protein, locates predominantly in the nucleus. It has been identified as a component of the PODs. During extracellular stimulation, Daxx could be recruited to the cytoplasm with the existence of Ask1. Therefore, it is a shuttle protein. Daxx should contain nuclear localization signal (NLS) and nuclear export signal (NES) motifs. To identify the NES and NLS motifs on Daxx, Daxx were truncated into four segments. Several amino acids on the predicted NES and NLS motifs were mutated. Our results showed that the truncated Daxx fragments D1 (containing NES) and D4 (containing NLS2) could be translocated into nucleus independently. However, either NES or NLS2 mutants disrupted their translocation into nucleus. It indicated that both NES and NLS2 motif of Daxx were involved in the nuclear transport. Nevertheless the co-transfection of SUMOs and Daxx showed that the interactions between SUMO active form and Daxx mutants and between inactive SUMO and Daxx wild type rescued the nuclear transport function of Daxx mutants and inactive SUMO. Therefore, SUMO may play a role in the nuclear transport of Daxx by either sumoylation or interaction with Daxx in cytoplasm, and Daxx may recruit inactive SUMOs into nucleus by interaction.

Abstract Human ubiquitin-like SUMO-1/2/3 proteins have been identified. The 3-D structure of the SUMO-1 has been shown to be very similar to that of ubiquitin, although their sequences share only 18 % identity. Unlike ubiquitination targets proteins for degradation, sumoylation appears to regulate a number of cellular processes such as protein-protein interaction, subcellular localization, protein stability, apoptosis, cell cycle and so on . Our laboratory has cloned cDNAs encoding human SUMO-2, mouse SUMO-2 and SUMO-3, as well as a single SUMO gene from nematode and Drosophila. Recently (Su & Li, Gene 296:65-73,2002), Su & Li have performed data-mining on current human genomic sequence and found the presence of only three SUMO-1/2/3 functional genes located at chromosome no. 2q33, 17q25.1 and 21q22.3, respectively, as well as eight SUMO-1 pseudogenes and 23 SUMO-2 pseudogenes. The protein-coding sequence of SUMO-1 gene is interrupted by four introns , while those of SUMO-2/3 genes are interrupted by only three introns. In this study , most of SUMO-1/2/3 proteins were show to be localized on nuclear membrane, nuclear bodies and cytoplasm, respectively. The N-terminus-deleted SUMO-1 proteins was further shown to be localized on nuclear membrane and in cytosol, while the mutant SUMO-2/3 proteins were localized only in the cytosol. The inactive precursor form of SUMO-3 was exclusively localized in the cytosol. The activation of SUMO-3 in HeLa cells was triggered by actinomycin D and its location was shifted from cytosol to nucleus. Further, the inactive precursor of SUMO-3 was reduced in HeLa cells treated with nocodazole and arsenic trioxide.

The 2-micron circle of Saccharomyces cerevisiae is one of a small number of similar DNA plasmids found only in budding yeast. To understand how this cryptic parasite persists, despite conferring no advantage to the host, I investigated the plasmid-encoded Rep1 and Rep2 proteins. Interaction of Rep1 and Rep2 with each other and with the plasmid STB locus is required for equal partitioning of plasmid copies at mitosis. The Rep proteins also repress expression of Flp, the recombinase that mediates plasmid copy-number amplification. In this study, absence of Rep1 and Rep2, or over-expression of the plasmid-encoded Raf antirepressor, increased expression of a longer, novel FLP transcript. Translation of this mRNA may explain elevated Flp activity at low plasmid copy number. Raf competed for Rep2 selfassociation and interaction with Rep1, suggesting the mechanism of Raf anti-repression. Deletion analysis identified a target site for Rep protein repression of FLP that is also repeated in the STB locus, suggesting this as the sequence required for Rep protein association with both regions of the plasmid. Distinct roles for Rep1 and Rep2 were identified; Rep1 was found to depend on Rep2 for post-translational stability, with Rep2 dependent on Rep1 for stable association with STB. Lysine-to-arginine substitutions in Rep1 and Rep2 impaired their association with the host covalent-modifier protein SUMO, suggesting these were sites of sumoylation. The substitutions did not affect interaction of the Rep proteins with each other or their stability but did perturb plasmid inheritance, suggesting that Rep protein sumoylation contributes to their plasmid partitioning function. When Rep1 was mutant, both Rep proteins lost their normal localization to the nuclear foci where 2-micron plasmids cluster, and were impaired for association with STB, supporting this as the cause of defective plasmid inheritance. The potential sumoylation-dependent association of the Rep proteins with the 2-micron plasmid partitioning locus suggests the plasmid has acquired a strategy common to eukaryotic viral and host genomes that depend on sumoylation of their segregation proteins for faithful inheritance. Collectively, my results shed light on how the 2-micron plasmid maintains the delicate balance of persisting without harming its host.

This diploma thesis aims to study VANET (vehicular ad hoc network), to describe the theory of this networks and describe attributes of these networks and to set the starting point for practical part. Thesis includes VANETs possibilities, its signal transportation and description of routing protocols. Another goal was to familiarize program NS-3 and set up simulation models in its interface. The main output of the practical part is program which simulates vehicle movement in Brno city and set the communication module which is adapted to fulfill the most realistic transmission capabilities. Practical part is divided to two parts. The goal of the first one was to simulate basic communication in theorized unrealistic scenario and the second part was the more realistic scenario. Overall, throughout the practical part was tested a variety of attributes, such as mobility models, standards, routing protocols and other parameters that provided diversity in final results. All the results, which consisted of summary of basic transmission capabilities and reclassification of the applicability of those technologies in real world, are discussed in the summary of the simulations output.

This paper describes a partitioning algorithm for real-world transportation networks incorporating previously unaccounted parameters like signalized traffic intersection, road segment length, traffic density, number of lanes and inter-partition communication overhead due to the migration of vehicles from one partition to another. We also describe our hypothetical framework for distributed simulation of the partitioned road network on SUMO, where a master controller is currently under development using TraCI APIs and MPI library to coordinate the parallel simulation and synchronization between the sub-networks generated by our proposed algorithm.