1 Viruses are submicroscopic obligate parasites that depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. In the recent times, many new RNA viruses have evolved that possess the potential threat to plants and animals. One among them is Tospovirus (Family Bunyaviridae) which has severely affected the agricultural productivity in India. One of the Tospoviruses GBNV is a major challenge of crop production in south India. Tospoviruses shares several features such as morphology, genome structure and organization with members of other genera in the family Bunyaviridae. Virus particles are 80–120 nm in diameter. The genome includes three RNAs referred to as large (L), medium (M) and small (S). The L RNA is in negative-sense while the M and S RNAs are ambisense. The L RNA codes for the RNA-dependent RNA polymerase (RdRp), and the M RNA for the precursor of two glycoproteins (GN and GC) and a non-structural protein (NSm). The S RNA codes for the N protein and another non-structural protein (NSs). Tospovirus infection is an emerging threat for agricultural productivity in India. Therefore, biochemical and molecular characterization of these viruses is essential for developing various strategies for control of these diseases. 2 Present thesis deals with biochemical characterization of nonstructural protein, NSs of GBNV. 3 A review of literature on Tospovirus genome organization, replication, transcription, translation and assembly is presented in Chapter I. This chapter also includes the recent work on all the proteins encoded by the tospoviruses. 4 The objectives of the present study are as follows; a. Cloning, expression, purification and biophysical characterizations of rNSs. b. Analysis of its NTPase/dATPase activity c. Demonstration of nucleic acid 5’ phosphatase activity d. Characterization of nucleic acid unwinding activity of rNSs 5 The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in bacteria, protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology are described in chapter II 6 NSs of TSWV was shown to be suppressor of gene silencing (PTGS) in 2002. Since then there has been no further work on this protein. Till date neither in vitro nor in vivo study of NSs of any tospovirus has been carried out in detail. To gain insight into the biochemical function of rNSs, the NSS gene was cloned, overexpressed in E.coli and purified. The NSS gene, was cloned into pRSET-C vector. 7. Chapter 3 deals with cloning, overexpression, purification and biophysical characterization of GBNV NSs in terms of secondary structure analysis as well as its interaction with siRNA and ssRNA. The results provide the evidence that rNSs was successfully expressed in E.coli and purified (Fig. 3.1). Molecular mass of purified rNSs was confirmed by MALDI TOF, which gave the molecular mass of expected size 51.5 kDa (Fig. 3.2) Circular dichroism study revealed that rNSs has negative ellipticity peak at 215 and 223 nm typical of a globular protein. The protein had an emission maximum at 340 nm (Fig 3.3 B) when exited at 280 nm, which reflects that rNSs is well folded. Thermal melting study (Fig 3.3 C) showed rNSs had a reasonably high Tm (65°C). So overall, spectral study suggested that purified rNSs was soluble, well folded and thermally stable and could be used for further biochemical assay. The oligomeric status of the protein was determined by size exclusion chromatography to be trimeric (156 kDa, Fig 3.5). Purified rNSs was used to raise the polyclonal antibodies in rabbit. The antiserum could detect rNSs specific band only in IPTG induced sample not in uninduced sample (Fig 3.6). 50% binding was observed at 100 ng/ml of antigen showing that these antibodies were of high affinity (Fig 3.7 B). Further, the 50% binding was observed at 1:34000 dilution of the antiserum, which suggests that high titer antibodies against rNSs were obtained (Fig 3.7 A). 8 Further, the RNA binding property of rNSs was examined. Synthetic 21 bp siRNA and in vitro transcribed 100 nt ssRNA was used to analyze the RNA binding property of rNSs. Indeed rNSs was able to bind with 100 nt ssRNA (Fig 3.8 A) or 21 nt siRNA in a protein concentration dependent manner (Fig 3.8 B). The binding however did not require presence of divalent cation such as Mg 2+ (Fig 3.8 C). In order to understand the biological function of rNSs, its interaction with the structural protein, NP by ELISA was investigated. rNSs could interact with the NP protein (Fig 3.9) . Further 15 amino deletions from C terminus of NP did not affect its interaction with rNSs protein (Fig 3.9), which suggest that the C terminal 15 amino acid residues of NP are not essential for interaction with rNSs in vitro. 9. Sequence analysis of GBNV NSs revealed the presence of Walker motifs A (GxxxxGKT) and B (DExx) in its primary structure (Fig 4.2). The proteins that possess the Walker motifs A and B exhibit ATPase activity. Therefore, the purified rNSs was tested for its ability to hydrolyze ATP in the absence and presence of poly(A) (chapter IV). rNSs could hydrolyze [γ-32P] ATP in a concentration-dependent manner (Fig. 4.3 A). Further, ATPase activity was stimulated in presence of poly(A) (Fig. 4.3 B). Quantitative analysis of reaction product suggested that the reaction was linear in the presence of poly(A) upto 1.6 µg of rNSs (Fig. 4.3 C). 10. The product of ATP hydrolysis by rNSs had the same mobility as the phosphate released by RecoP51 ATPase, a positive control used in the assay. In contrast, another viral protein from the Cotton leaf curl virus, His tagged-AV2, purified in same way as rNSs, did not show the release of phosphate, suggesting that the activity was not due to the histidine tag present at the N-terminus of rNSs. Further, no release of phosphate could be seen when immunodepleted rNSs was used suggesting that the activity was inherent to the protein and was not due to bacterial contamination (Fig 4.3 lane 7). Time course analysis of ATPase activity revealed that the reaction is linear up to 25 mins (Fig 4.4). Further, pH profile was a typical bell shaped curve with a distinct pH optimum at pH 7.0 (Fig 4.5 A) and the temperature optimum was at 25 °C(Fig 4.5 B). Most of the known viral ATPases require the divalent cation for their activity. The rNSs exhibited the optimum ATPase activity between 2-2.5 mM of MgCl2. The reaction was inhibited by increasing concentration of EDTA demonstrating the requirement of Mg2+ for ATP hydrolysis (Fig. 4.7). Further, the ATPase activity of rNSs was inhibited by increasing concentrations of non-hydrolyzable analog of ATP (Fig. 4.8) and was not inhibited by AMP (Fig 4.9) suggesting that rNSs is not a nucleotidyl phosphatase and is a true ATPase. Limited proteolysis of rNSs suggested that core domain was 23 kDa in size and could catalyze ATP hydrolysis (Fig. 21 and 4.22). 11. Interestingly rNSs not only cleaved ATP rather it could hydrolyze all rNTPs as well as dATP (Fig 4.10). Kinetic parameters were determined for its enzymatic activity. Comparison of the kinetic constants of rNSs NTPase activity revealed little variation, suggesting that the rNSs has a broad substrate specificity (Fig 4.10- 4.15 and table 4.1). 12. To assess the role of amino acids in Walker motif A and B (Fig. 4.16) site specific mutants K189A and D159A were generated ( Fig 4.17) confirmed by sequencing, overexpressed in E.coli and purified (Fig. 4.18). Point mutation in Walker motif B (D159A) reduced the ATPase activity (Fig 4.19) where as point mutation in Walker motif A (K189A abolishes the activity (Fig 4.19). 13. Chapter V deals with the nucleic acid 5’ phosphatase activity of rNSs. Experimental evidence presented in this chapter clearly shows that rNSs can cleave the single phosphate from the ssDNA, ssRNA, dsRNA and dsDNA. Nucleic acid 5’ phosphatase activity of rNSs was inhibited by AMP and ATP (Fig 5.2 and Fig 5.3). Interestingly the K189A mutant rNSs was as active as wild type rNSs where as D159A mutant showed slightly reduced activity (Fig 5.7 C). 14. As mentioned earlier, rNSs was shown to possesses the RNA stimulated NTPase/dATPase activity, a hallmark of all known helicases. Therefore, its nucleic acid unwinding activity was examined using dsDNA and dsRNA as a substrate. rNSs was able to unwind the dsDNA as well as dsRNA in a ATP dependent manner (chapter VI, Fig. 6.1 and 6.5 respectively). ATP and Mg2+ are essential cofactors for the unwinding activity (Fig. 6.1). While the unwinding activity could be observed with ATP and to some extent with dATP, all other NTPs and dNTPs failed to support the helicase function of rNSs (Fig 6.2) Further experimental evidence suggested that rNSs is a bidirectional helicase (Fig. 6.3). D159A mutation in Walker motif B resulted in reduced helicase activity where as K189A mutation in walker Motif A completely abolished the DNA as well as RNA helicase activity of rNSs (Fig. 6.6 and Fig 6.7 respectively). Therefore, mutational analysis clearly suggests that helicase activity is an intrinsic property of rNSs. 15. In conclusion rNSs of GBNV is multifunctional enzyme. This is the first report on the demonstration that rNSs is an non canonical ATP dependent helicase in the Bunyaviridae family. In addition to being a suppressor of PTGS, NSs may also regulate the viral replication and transcription by modulating the secondary structure of the viral genome. This new research finding on NSs might pave way for further studies on its role in viral replication and transcription.

2003 invaderade USA tillsammans med ett antal av dess allierade Irak. Invasionen genomfördes utan mandat från FN:s säkerhetsråd. Uppsatsen handlar om hur USA och Bushadministrationen motiverade den invasionen och om hur FN:s och FN-stadgans regelverk reglerar internationell våldsanvändning.Syfte med uppsatsen är att se hur Bushadministrationens motiveringar förhåller sig till FN-stadgan och till den tillhörande resolutioner.Detta har gjorts genom en kvalitativ textanalys där tal och officiella dokument analyserats. Som teoretisk grund har realismen använts. Realismen har även använts som en förklaringsmodell för varför USA handlade som de gjorde.Resultatet i uppsatsen visar att USA:s invasion av Irak 2003 ej var förenlig med FN-stadgan och till den tillhörande resolutioner. Men tydliga kopplingar mellan USA:s aggerande och Realismen kan identifieras.

Tato práce se zabývá návrhem a implementací řešení pro jednotnou síťovou autentizaci pro operační systém Linux založeného na integraci systémových démonů WinBind a SSSD. Cílem je navrhnout takové řešení, které umožní autentizaci Linuxových klientů do domén spravovaných adresářovými službami Windows Active Directory a domén spravovaných adresářovými službami dostupnými na Linuxu současně. První dvě kapitoly seznámí čtenáře s autentizačními mechanizmy a technologiemi, které se pro tyto účely použivají na operačních systémech Windows a Linux. Třetí kapitola se zabývá jádrem práce a vysvětluje rozhodnutí učiněná při návrhu implementovaného řešení. Samotná implementace je pak popsána v kapitole čtyři. Poslední kapitoly popisují experimenty a testování pro vybrané případy užití s návrhy a popisem možných rozšíření do budoucna.

MA (Political Science) / Department of Development Studies / See the attached abstract below

No description available.

Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.

Tato práce se v první části zabývá představením technologií a nástrojů pro centrální správu a přihlašování uživatelů v GNU/Linux. Ukazuje využití adresářových služeb v síťové infrastruktuře, konkrétně služby NIS a její porovnání s dnes pravděpodobně nejrozšířenější adresářovou službou LDAP. Dále práce popisuje proces autentizace na klientských stanicích, konkrétně použití PAM a NSS a možné rozšíření celého systému zavedením cache díky démonu SSSD. Druhá část popisuje návrh a implementaci NIS provideru pro SSSD.

This thesis submits broader understanding of George W. Bush and Barack Obama presidencies in terms of their Middle Eastern policy, in particular the issue of Iranian nuclear policy. The main interest lies in an intention to conclude, which particular circumstances led the American governments to rethink their position towards the Iranian nuclear program, bearing in mind changes which took place in security, economic and foreign policy interests of the United States. The first chapter describes the theoretical framework upon which the entire thesis is based upon. The second chapter provides a short review on the MENA region, describing the most visible factors and issues form the very last years of the second administration of Barack Obama. The third chapter is based upon the research of the Iranian position in the MENA region, its potential, and what possibilities the U.S. government posses in order to contain Iran from spreading its influence. The next chapter deals with an issue of nuclear terrorism and its connections towards Iran, evaluation of possible nuclear terrorist menace and possibilities of so called "Dirty Bombs" usage. The fifth chapter is based upon the research of the first presidency of George W. Bush and the policies which were implemented after the terrorist attacks on 11...

The 2002 National Security Strategy of the US (NSS 2002) appeared to have presented a momentous approach to self-defense. To many, the doctrine of preemptive selfdefense seemed to challenge the legal and political foundations of the post-World War II international order. Some saw in the US stated reliance on preemption a direct threat to the international system embodied in the UN Charter. The prima facie case that the US position was novel and even dangerous appeared persuasive. This thesis attempts to assess the exceptionality of NSS 2002 in its formulation and implications. This question of exceptionality is broadly divided into two sections. The first section deals with internal exceptionality, in terms of means (the deliberation and drafting processes) and ends (the US defense posture). The second section deals with external exceptionality in the broader terms of possible consequences outside the US. Section One begins by establishing the grounds for looking into the formulation of NSS 2002, and provides the background for that Strategy's mandated precursors. After exploring how National Security Strategy documents are conceived and framed, Section One discusses the Strategy as it was published, and examines a sampling of contemporaneous reactions to its publication. Section Two concentrates on the second part of the research question, and utilizes a thematic approach - in terms of the use of force, the international security environment, and international law. Possible consequences of the proposed US response to contemporary security challenges are considered in these three key areas.

The 2002 National Security Strategy of the US (NSS 2002) appeared to have presented a momentous approach to self-defense. To many, the doctrine of preemptive selfdefense seemed to challenge the legal and political foundations of the post-World War II international order. Some saw in the US stated reliance on preemption a direct threat to the international system embodied in the UN Charter. The prima facie case that the US position was novel and even dangerous appeared persuasive. This thesis attempts to assess the exceptionality of NSS 2002 in its formulation and implications. This question of exceptionality is broadly divided into two sections. The first section deals with internal exceptionality, in terms of means (the deliberation and drafting processes) and ends (the US defense posture). The second section deals with external exceptionality in the broader terms of possible consequences outside the US. Section One begins by establishing the grounds for looking into the formulation of NSS 2002, and provides the background for that Strategy's mandated precursors. After exploring how National Security Strategy documents are conceived and framed, Section One discusses the Strategy as it was published, and examines a sampling of contemporaneous reactions to its publication. Section Two concentrates on the second part of the research question, and utilizes a thematic approach ¿ in terms of the use of force, the international security environment, and international law. Possible consequences of the proposed US response to contemporary security challenges are considered in these three key areas.