Dissertations / Theses on the topic 'Ns3'

A dengue é uma doença infecciosa com grandes taxas de morbimortalidade, causada pelo vírus da dengue (DENV). Segundo a Organização Mundial de Saúde, cerca de 50 a 100 milhões de pessoas são infectadas anualmente em mais de 100 países tropicais e subtropicais de todos os continentes. O espectro clínico da infecção pelo DENV pode incluir formas assintomáticas ou sintomaticas que variam desde uma febre indeterminada e autolimitada, passando pela febre clássica da dengue (FD) até quadros graves denominados febre hemorrágica da dengue/síndrome do choque da dengue (FHD/SCD). Recentemente, ocorreu um dramático aumento do número de casos de FHD/SCD nas Américas, e este aumento coincidiu com a introdução do dengue sorotipo 3, genótipo III. No presente trabalho, objetivou-se a clonagem e a expressão das proteínas NS1 e NS3 do vírus da dengue tipo 3. As proteínas NS1 e NS3 do DENV-3 foram clonadas e expressas com sucesso em sistema procarioto. A amplificação dos genes das proteínas NS1 e NS3 foi realizada por RT-PCR, o qual gerou amplicons de cerca de 1050 e 1850 pb, respectivamente. Em seguida, os genes foram clonados por inserção dos amplicons no vetor plasmidial pCR-XL. Os genes de NS1 e NS3 foram subclonados no vetor de expressão pQE-30 através de sítios de restrição para as enzimas BamHI e HindIII. A expressão proteica foi obtida em sistema procarioto utilizando a cepa BL21(DE3) de E. coli, resultando em proteínas de 45 e 70 kDa as quais foram confirmadas por análises em Western blot utilizando como anticorpo primário fluido ascítico imune de camundongos e soro de pacientes com dengue. Estas proteínas virais podem ser utilizadas para estudos relacionados à patogênese, replicação e mecanismos de escape do sistema imune do DENV, além disso, podem ser potencias antígenos em métodos de diagnóstico.
Dengue is an infectious disease with high morbidity and mortality rates caused by dengue virus (DENV). According to the World Health Organization, about 50 to 100 million people are infected annually in more than 100 tropical and subtropical countries from all continents. The clinical spectrum of DENV infection can includes asymptomatic or symptomatic forms ranging from undetermined and self-limited fever, through dengue fever (DF) to severe disease called dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Recently, there has been a dramatic increase in the number of cases of DHF/DSS in the Americas, and this increase coincided with the introduction of dengue virus type 3 (DENV-3), genotype III. The present study aimed to clone and express NS1 and NS3 proteins of DENV-3. The NS1 and NS3 proteins of DENV-3 was successfully cloned and expressed in a prokaryotic system. Amplification of NS1 and NS3 genes was carried out by RT-PCR, which yielded amplicons of approximately 1050 and 1850 bp, respectively. Then, the genes were cloned by inserting the amplicons into the plasmid vector pCR-XL. NS1 and NS3 genes were subcloned into the expression vector pQE-30 through the restriction sites for BamHI and HindIII enzymes. The protein expression was obtained in a prokaryotic system using the strain BL21 (DE3) of E. coli, resulting in 45 and 70 kDa proteins, which were confirmed by Western blot analysis using immune mouse ascitic fluid and serum of patients with dengue as primary antibody. These viral proteins can be used to study the pathogenesis, mechanisms of replication and immune escape of DENV, moreover, can be potential antigens in diagnostic methods.

Cílem bylo navrhnout dva simulační scénáře v prostředí NS-3. První scénář obsahuje ARQ (Automatic Repeat Request) metody v TCP (Transmission Control Protocol). Je v něm porovnání Stop-and-Wait, Go-Back-N a Selective-Repeat metod. Teoretická část obsahuje TCP a ARQ. Druhý scénář je o způsobech přenosu zpráv. Vytvořený scénář převážně s komutací paketů a buněk a teoretické základy jsou obsaženy v práci. Je v něm porovnání metod komutací s různou velikostí paketu/buňky, počtem uzlů a důsledek zpoždění v každé metodě. Scénáře jsou ve formě laboratorní úlohy s instrukcemi k vypracování.

Les Flavivirus transmis par les moustiques comme le virus du Nil occidental, le virus de la dengue, le virus de la fièvre jaune, le virus de l’encéphalite japonaise et le virus Zika constituent des préoccupations croissantes de santé publique. Ils se sont répandus dans le monde au cours des dernières décennies, et les épidémies sont devenues plus fréquentes et plus sévères. Chaque année, des millions de personnes sont infectées et environ 50 000 patients décèdent d’infections à Flavivirus. Malgré les nombreux efforts de recherche, il n’y a actuellement aucun médicament antiviral spécifique disponible, et des nouvelles stratégies antivirales sont indispensables. Comprendre comment les Flavivirus fonctionnent au niveau moléculaire aidera à découvrir des nouvelles cibles pour l'intervention thérapeutique. Les Flavivirus ont un génome d'ARN simple brin de polarité positive qui code pour trois protéines structurales et huit protéines non structurales. Seules deux des huit protéines non structurales ont des activités enzymatiques. NS3 possède un domaine protéase et un domaine hélicase, et NS5 a un domaine méthyl- et guanylyltransférase et un domaine ARN polymérase ARN-dépendante. Ensemble, ils répliquent le génome viral. Ici, nous caractérisons l'interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive. Un modèle d'interaction comprenant NS3, NS5 et l’ARN viral a été développé basé sur des structures cristallines connues ainsi que des activités enzymatiques des deux protéines individuelles, et ce modèle a été soumis à des simulations de dynamique moléculaire. Les interactions potentielles entre les protéines NS3 et NS5 ont été identifiées. Les résidus impliqués dans ces interactions ont été mutés dans un réplicon du virus du Nil occidental et les effets de ces mutations sur la réplication virale ont été évalués. Une région particulière à la surface de la protéine NS3 a été identifiée comme étant cruciale pour la réplication virale, très probablement parce qu'elle interagit avec NS5. Cette région pourrait être une cible attrayante pour la recherche de composés qui pourraient interférer avec l'interaction entre NS3 et NS5 et donc posséder un potentiel antiviral intéressant.
Abstract : Mosquito-borne Flaviviruses like West Nile virus, Dengue virus, Yellow Fever virus, Japanese encephalitis virus, and Zika virus are increasing public health concerns. They have spread globally during the past decades, and outbreaks have recently become more frequent and more severe. Every year, millions of people are infected, and approximately 50,000 patients die from Flavivirus infections. Despite extensive research efforts, there are currently no specific antiviral drugs available, and new antiviral strategies are greatly needed. Understanding how Flaviviruses work on a molecular level will help in uncovering new points for therapeutic intervention. Flaviviruses have a single-stranded RNA genome of positive polarity that encodes three structural and eight non-structural proteins. Only two of the eight non-structural proteins have enzymatic activities. NS3 has an N-terminal protease domain and a C-terminal helicase domain, and NS5 has an N-terminal capping enzyme domain and a C-terminal RNA-dependent RNA polymerase domain. Together, they replicate the viral genome. Here we characterize the NS3:NS5 interaction within the West Nile virus RNA replicase complex during positive strand synthesis. An interaction model including NS3, NS5 and viral RNA was developed based on the known crystal structures as well as enzymatic activities of the two individual proteins, and this model was subjected to molecular dynamics simulations. Potential interactions between the NS3 and NS5 proteins were identified. Residues involved in these interactions were mutated in a West Nile virus replicon, and the effects of these mutations on viral replication were evaluated. One particular region on the surface of the NS3 protein was identified to be crucial for viral replication, most likely because it mediates the interaction with NS5. This region might be an attractive target for the search of compounds that could interfere with the NS3:NS5 interaction and therefore possess an interesting antiviral potential.

As near-Earth space becomes more populated with large constellations of satellites and research into spacecraft autonomy and disaggregation becomes more prevalent, it will be increasingly important to design effective communication procedures between satellites to efficiently share resources and avoid collisions. Though there have been several space networking simulation tools created in recent years, they all lack rigorous astrodynamics models or use high-fidelity but bulky and computationally taxing commercial software. This research presents Spacecraft-ns3, an extension to the ns-3 network simulator. Using a modular approach, Spacecraft-ns3 propagates orbit state, plans discrete events, and analyzes network metrics and flows. A case study using Spacecraft-ns3 is presented for exploratory space network analysis.
Master of Science
Near-Earth space has become more crowded in recent years due to the increasing number of large constellations of satellites in this region. Autonomous vehicle research has been applied to Earth satellites primarily to share power and computing resources between satellites, or to prevent collisions between satellites. Both of these factors require effective communication procedures between satellites, which can be inexpensively simulated with network simulators. However, network simulators are primarily designed for ground-based use, and must be combined with an astrodynamics simulator to effectively simulate satellite networks. This research presents Spacecraft-ns3, an integrated simulator that defines spacecraft orbits and attitude, and analyzes network activity. This simulator improves upon prior simulation efforts by extending the ns-3 network simulator with efficient and high-fidelity astrodynamics models. The Spacecraft-ns3 simulator is demonstrated in an exploratory case study.

Diploma thesis provides an introduction to the ns-3 network simulator. It describes the basic abstactions and components of the instrument. This work further discusses the theory, which was applied during simulation creation. The concept of unicast, the issue of IP addressing and routing in networks are specificaly analyzed. The practical part is focused on the creation of four laboratory tasks within the scope of Advanced Communication Techniques. The first task is dedicated to the the issue of static unicast routing in the network. It clarifies the impact of metrics on the process of routing. The following task compares the different types of ICMPv4 and ICMPv6 messages. The third task discusses additional functions of the ICMPv6. Specifically, it focuses on five different types of ICMPv6 messages defined by NDP (Neighbor Discovery Protocol) and their role in the network traffic. The last task focuses on ensuring the quality of services (QoS) in wireless networks, by setting priorities and thereby dividing the traffic into different classes. Attention is focused on parameters such as throughput, delay and jitter.

This thesis deals with the powerline communication focusing on the simulations of channel transfer functions. The introduction summarizes basic information about PLC followed by the analysis of the available PLC Software simulator (implemented in NS-3) and the analysis of implemented noises and interferences and its extensions. One chapter is devoted to possibilities of link capacity calculation. Within the thesis, various methods of calculation of primary parameters of transmission line were implemented and the simulator was compared with other freely available simulators. Subsequently, simulations were carried out focused on channel transfer functions in different topologies, on the impedance changes of branch, on the lengths of branch, and changes in the direct path between communicating nodes.

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Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
O vírus da dengue (DENV) consiste de quatro sorotipos antigenicamente relacionados: DENV-1, DENV-2, DENV-3 e DENV-4. Apesar dos diversos esforços para o desenvolvimento de uma vacina contra dengue, ainda não há nenhuma comercialmente disponível. As proteínas não estruturais 1 e 3 (NS1 e NS3) são indicadas como antígenos promissores para o desenvolvimento de uma vacina contra DENV. Segundo alguns estudos, a proteína NS1 é capaz de induzir uma resposta protetora de anticorpos com atividade de fixação do complemento. A proteína NS3, que realiza reações enzimáticas essenciais para a replicação viral, parece ser imunogênica, contendo um predomínio de epítopos para linfócitos T CD4+ e CD8+. No presente trabalho nós avaliamos o potencial de vacinas de DNA baseadas nas proteínas NS1 e NS3 de DENV-2. Foram construídos cinco plasmídeos, pcTPANS3, pcTPANS3H, pcTPANS3P, pcTPANS3N e pcTPANS3C, contendo a seqüência que codifica o peptídeo sinal do ativador de plasminogênio de tecido humano (t-PA) fusionado ao gene NS3 inteiro ou partes destes. Todos estes plasmídeos mediaram a expressão das proteínas recombinantes in vitro em células eucarióticas Camundongos foram inoculados com estes plasmídeos e desafiados com DENV-2 por via intracerebral (i.c.). Nenhuma destas construções induziu níveis satisfatórios de proteção. Além dos plasmídeos com NS3, foram construídas quatro vacinas de DNA baseadas no gene NS1: 1 - pcENS1, que codifica a região C-terminal da proteína E fusionada à NS1, 2 - pcENS1ANC, similar ao pcENS1 com a adição da porção N-terminal da NS2A (ANC), 3 - pcTPANS1, que codifica o peptídeo sinal t-PA fusionado à NS1 e 4 - pcTPANS1ANC, semelhante ao pcTPANS1 com a adição da seqüência ANC. A proteína NS1 recombinante foi detectada nos extratos celulares e sobrenadante das culturas de células BHK transfectadas com pcTPANS1, pcENS1 e pcENS1ANC. Tais resultados indicam que as seqüências sinais t-PA e E direcionaram a NS1 para secreção. A proteína NS1 também foi observada associada à membrana plasmática de células transfectadas com pcENS1ANC, demonstrando a importância da seqüência ANC para o seu ancoramento. Todos os camundongos imunizados com pcTPANS1 ou pcENS1 produziram altos níveis de anticorpos, direcionados principalmente para epítopos conformacionais da NS1, enquanto que somente metade dos animais inoculados com pcENS1ANC apresentaram níveis detectáveis de anticorpos A resposta de anticorpos se mostrou duradoura (até 56 semanas após a primeira dose das vacinas), e os animais apresentaram uma rápida resposta secundária após um reforço de DNA. Camundongos imunizados com os plasmídeos pcTPANS1 e pcENS1 se mostraram protegidos contra desafios com DENV-2 por via i.c., sendo o pcTPANS1 levemente mais protetor. Estes dois plasmídeos ativaram a produção de diferentes subclasses de IgG específicas contra NS1. Não foi observada proteção interespecífica quando camundongos imunizados com pcTPANS1 foram desafiados por via i.c. com DENV-1. Os animais imunizados com o pcTPANS1 foram desafiados com DENV-2 por via intraperitoneal e também se mostraram protegidos. Neste modelo de desafio, foi observada uma diminuição dos efeitos histopatológicos do vírus no fígado dos animais vacinados. Resultados preliminares sugerem à lise de células infectadas com DENV-2, dependente do complemento, na presença dos anticorpos direcionados contra NS1
Dengue virus (DENV) consists of four antigenically related serotypes: DENV-1, DENV-2, DENV-3 and DENV-4. Although considerable research has been conducted towards the development of a DENV vaccine, no vaccine is yet commercially available. The non-structural proteins 1 and 3 (NS1 and NS3) have been identified as promising antigens for the development of vaccines against DENV. According to some reports, NS1 can elicit a protective antibody response with complement-fixing activities. NS3, a protein that carries out enzymatic reactions essential for viral replication, appears to be immunogenic, presenting a preponderance of the CD4+ and CD8+ T cell epitopes. In the present work we investigate the potential of DNA vaccines based on the DENV-2 NS1 and NS3 proteins. We constructed five recombinant plasmids, pcTPANS3, pcTPANS3H, pcTPANS3P, pcTPANS3N and pcTPANS3C, which contain the sequence that codes the signal peptide derived from the human tissue plasminogen activator (t-PA) fused to the full or partial length of the DENV-2 NS3 gene. Results indicated that these plasmids promoted the expression of recombinant proteins in eukaryotic cells. Mice were inoculated with these plasmids and challenged by the intracerebral (i.c.) route with DENV-2. None of these constructs induced acceptable protection. Moreover, we constructed four DNA vaccines based on the DENV-2 NS1 gene: 1 - pcENS1, coding the C-terminal of the E protein fused to NS1, 2 - pcENS1ANC, similar to pcENS1 with the addition of the N-terminal of NS2A (ANC), 3 - pcTPANS1, coding the t-PA signal sequence fused to NS1 and 4 - pcTPANS1ANC, similar to pcTPANS1 with the addition of the ANC sequence. The recombinant NS1 protein was detected in cell extracts and culture supernatants from pcTPANS1-, pcENS1- and pcENS1ANC-transfected BHK cells. Such results indicated that the E and t-PA sequences targeted NS1 to secretion. NS1 was also observed in association with plasma membrane of pcENS1ANC-transfected cells, which demonstrated the importance of the ANC sequence for cell anchoring. High levels of antibodies, mainly recognizing surface-exposed conformational epitopes of NS1, were induced in all mice immunized with pcTPANS1 and pcENS1, while only half of pcENS1ANC-inoculated animals presented detectable antibody levels. Long-term antibody response was observed in pcTPANS1 and pcENS1 immunized animals (56 weeks after the first vaccine inoculation) and there was a rapid secondary response after a DNA booster. Protection was elicited in pcTPANS1- and pcENS1-immunized mice challenged with DENV- 2 by the i.c. route and the pcTPANS1 seemed to generate a slightly higher protection. Moreover, these two plasmids induced different NS1-specific IgG subclasses. No protection was displayed when pcTPANS1-immunized animals were i.c. challenged with DENV-1. Animals inoculated with pcTPANS1 were also protected when they were challenged with DENV-2 by the intraperitoneal route. Liver tissue from vaccinated animals presented a remarkable decrease of hepatic damages in this challenge mouse model. Preliminary results suggested the complement-mediated lyses of DENV-2 infected cells in the presence of the NS1-specific antibody.

Au cours de ma thèse je me suis intéressée aux interactions protéine-protéine (PPI’s). Les PPI’s jouent un rôle majeur dans une grande diversité de processus cellulaires et sont maintenant considérées comme une cible majeure dans le but de développer de nouveaux médicaments. Cependant, cibler ce type d’interactions requiert le développement de chimiothèques dédiées, permettant d’accélérer la découverte de molécules « touches ». Pour surmonter ce problème, une chimiothèque orientée PPI (2P2I3D) a été conçu au laboratoire. Dans un premier temps, j’ai donc évalué cette chimiothèque sur différents complexes possédant des interfaces variées. Les résultats obtenus ont révélé des taux de touches supérieurs à ceux obtenus avec des chimiothèques non orientées, de 0,2 à 1,6% contre 0,01 à 0,1%, respectivement. Cette étude a permis d’établir une preuve de concept de la faisabilité de créer une chimiothèque orientée PPI, permettant ainsi une accélération de la découverte de composés biologiquement actifs.Dans un deuxième temps, je me suis intéressée à l’interaction entre deux protéines majeures du virus de la dengue : les protéines NS3 et NS5. J’ai tout d’abord identifié et caractérisé un nouveau site d’interaction, ce qui m’a permis de mettre en évidence que cette interaction avait pour conséquence d’augmenter l’activité enzymatique du domaine hélicase. J’ai par la suite recherché et identifié des petites molécules chimiques capable d’inhiber cette interaction. Les différentes caractérisations effectuées ont permis de mettre en évidence un effet antiviral. Ces inhibiteurs constituent un excellent point de départ afin d’étudier plus en détail le rôle biologique de ce complexe
In my thesis I became interested in protein-protein interactions (PPI's). PPI's play a major role in a variety of cellular processes and are now considered a major target in order to develop new drugs. However, targeting such interactions requires the development of dedicated libraries, to accelerate the discovery of “hits”molecules .To overcome this issue, a focused chemical library PPI (2P2I3D) was designed in the laboratory.At first, I evaluated this chemical library on different complexes with diverse interfaces. The results showed higher hit rate to those obtained with non-oriented libraries, from 0.2 to 1.6% against 0.01 to 0.1%, respectively. This study has established a proof of concept of the feasibility of creating a focused chemical library PPI, thus accelerating the discovery of biologically active compounds.Secondly, I am interested in the interaction between two major proteins of dengue virus: the NS3 and NS5 proteins. I initially identified and characterized a novel interaction site, which allowed me to demonstrate that this interaction had the effect of increasing the enzymatic activity of the helicase domain. I searched and identified small molecules able to inhibit this interaction. The different characterizations helped to highlight an antiviral effect. These inhibitors are an excellent starting point to further explore the biological role of this complex

This thesis deals with the topic of network slicing technology in 5G networks, mainly on the RAN part. In the theoretical part, basic principles of 5G network slicing in core network part and RAN part are presented. Practical part contains a simulation scenario created in NS3 simulator with LENA 5G module. Results of this simulation are presented and discussed with the emphasis on RAN slicing.

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A Hepatite C é caracterizada por uma infecção crônica e progressiva causada pelo vírus da HCV. A proteína NS3 do vírus está envolvida no seu ciclo replicativo, sendo assim importante alvo de ação de antivirais denominados de ação direta. O inibidor da protease Boceprevir faz parte da primeira geração dessa classe de drogas. Porém, muitos pacientes apresentam resposta virológica não sustentada (RVNS) durante o tratamento devido à incapacidade de supressão da replicação viral. O objetivo desse trabalho foi avaliar a presença de mutações e polimorfismos genéticos na região codificadora da proteína NS3 do HCV em pacientes que apresentam indicação de uso do fármaco Boceprevir que estão associadas com à (RVNS). Amostras de 25 pacientes foram utilizadas para o sequenciamento da proteína NS3 do HCV e avaliadas de acordo com suas mutações através de análise filogenética e de modelagem molecular, mais especificamente, índice de reatividade e simulação de dinâmica molecular. A reconstrução filogenética e as análises de pressão de seleção mostraram que pacientes que apresentaram falha ou recidiva possuem vírus com algumas mutações em regiões na estrutura viral que são próximas aos sítios de ligação com o fármaco Boceprevir. As análises de índice de reatividade mostraram que a probabilidade da ligação covalente ocorrer entre ligante e receptor está associada com a probabilidade de desprotonação do aminoácido S139 da proteína NS3. Já os resultados de dinâmica molecular mostram que a estabilidade da interação fármaco e proteína não é alterada após mutações singulares, sugerindo que o mecanismo de resistência do HCV pode estar associado a uma combinação de mutações ou ainda a aspectos moleculares não analisados no presente trabalho, como mecanismos de acoplamento receptor e ligante e modos de vibração da proteína.
Hepatitis C is characterized by a chronic and progressive infection caused by the HCV virus. The NS3 protein of the virus is involved in its replicative cycle, thus being an important target for the action of so-called direct-acting antivirals. The protease inhibitor Boceprevir is part of the first generation of this class of drugs. However, many patients present unsupported virological response (UVR) during treatment because of inability to suppress viral replication. The objective of this work was to evaluate the presence of mutations and genetic polymorphisms in the coding of the VHC NS3 protein in patients with indication of use of the drug Boceprevir that are associated with unsustained virological response. Samples from 25 patients were used for the sequencing of the VHC NS3 protein and evaluated according to their mutations through phylogenetic analysis and molecular modeling, more specifically, reactivity index and molecular dynamics simulation. Phylogenetic reconstruction and selection pressure analyzes have shown that patients who have failed or relapse have viruses with some mutations in regions in the NS3 protein sequence that are close to the drug binding sites Boceprevir. Reactivity index analyzes have shown that the probability of covalent binding between ligand and receptor occurring is associated with the probability of deprotonation of the S139 amino acid of the NS3 protein. The molecular dynamics results show that the stability of the drug and protein interaction is not altered after single mutations, suggesting that the mechanism of resistance of HCV may be associated with a combination of mutations or molecular aspects not analyzed in the present work, as Receptor and ligand coupling mechanisms and modes of protein vibration.

Orientador: Rafael Plana Simões
Resumo: A Hepatite C é caracterizada por uma infecção crônica e progressiva causada pelo vírus da HCV. A proteína NS3 do vírus está envolvida no seu ciclo replicativo, sendo assim importante alvo de ação de antivirais denominados de ação direta. O inibidor da protease Boceprevir faz parte da primeira geração dessa classe de drogas. Porém, muitos pacientes apresentam resposta virológica não sustentada (RVNS) durante o tratamento devido à incapacidade de supressão da replicação viral. O objetivo desse trabalho foi avaliar a presença de mutações e polimorfismos genéticos na região codificadora da proteína NS3 do HCV em pacientes que apresentam indicação de uso do fármaco Boceprevir que estão associadas com à (RVNS). Amostras de 25 pacientes foram utilizadas para o sequenciamento da proteína NS3 do HCV e avaliadas de acordo com suas mutações através de análise filogenética e de modelagem molecular, mais especificamente, índice de reatividade e simulação de dinâmica molecular. A reconstrução filogenética e as análises de pressão de seleção mostraram que pacientes que apresentaram falha ou recidiva possuem vírus com algumas mutações em regiões na estrutura viral que são próximas aos sítios de ligação com o fármaco Boceprevir. As análises de índice de reatividade mostraram que a probabilidade da ligação covalente ocorrer entre ligante e receptor está associada com a probabilidade de desprotonação do aminoácido S139 da proteína NS3. Já os resultados de dinâmica molecular mostram que a estabi... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

This master thesis deals with creating laboratory exercises for students in simulator NS-3, where the IPv4 and IPv6 protocols, unicast and multicast transmission types, quality of services in wireless networks, furthermore protocols TCP and UDP are analyzed. The first section describes the NS-3 network simulator, the Eclipse development environment and the method how to connect them. In the next section protocols IPv4 and IPv6 are presented, the structure of the headers and the technique of fragmentation are described in detail. Based on this a laboratory exercise is created which deals with comparison of protocols IPv4 and IPv6. In the next section the types of transmissions are described, furthermore the multicast transmission type is explained more in detail. In the next practical section the second laboratory exercise is created, which deals with comparison of unicast and multicast transmission types. The subject of the following section is ensuring quality of services in data networks, the EDCA access method is discussed more in detail. In the next section the EDCA access method is implemented in a simulation as a laboratory exercise, in which the impact of the access category of EDCA to different transmission parameters is investigated. The next section deals with protocols TCP and UDP, furthermore with the structure of their headers. In the last section a laboratory exercise is created, in which the properties of the protocols TCP and UDP are compared.

La protéase NS3 est essentielle à la réplication du VHC et est l'une des cibles potentielles pour le développement de nouvelles thérapeutiques anti-VHC. Dans une première étude, la variabilité génétique de la protéase NS3 a été analysée pour 17 échantillons sériques pré-thérapeutiques issus de patients infectés par des VHC de génotype1 et répartis en fonction de leur réponse ultérieure à la bithérapie standard. Une certaine variabilité nucléotidique et protéique a été observée. La réponse ultérieure à la bithérapie et la variabilitéde la protéase virale n'étaient pas associées. Un profil particulier incluant 3 délétions et une insertion a été rapportée pour 4 variants issus de la quasi-espèce d'un patient. Dans une seconde étude, un profil génétique particulier en relation avec l'évolution de la cirrhose en CHC a été recherché. Aucun résidu ou motif spécifique n'a été détecté comme étant prédictif de l'évolution de la cirrhose chez une population cas/témoins de 10 patients cirrhotiques
NS3 protease is essential for HCV replication, and is one of the most promising targets for anti-HCV therapy. In a first work, the genetic heterogeneity of the protease gene was analysed in 17 HCV gentype 1 pre-therapeutic samples distributed according to their subsequent response to standard combination therapy. Variability of both nucleotide and amino acid sequences was found. No association between the outcome of bitherapy and mutational pattern before treatment was found. A particular pattern including three deletions and one insertion in four clones of the quasispecies was found for one patient. In a second work, as there are different arguments for a putative role of the HCV NS3 protease in the carcinogenesis process, we searched for a genetic pattern of NS3 protease in relation with evolution from viral cirrhosis to HCC. No specific residues or motifs were detected as predictive of cirrhosis outcome in a case controle population of 10 cirrhotic patients

Worldwide between 200 - 300 million people are chronically infected with the Hepatitis C Virus (HCV). For up to 20% of infected patients, chronicity can lead to cirrhosis and hepato-cellular carcinomas. HCV is a member of the Flaviviridae family, such as Dengue virus (DENV) and West Nile Virus (WNV), which has been classified into its own, Hepacivirus genus due to major differences in genomic organization and amino acid sequences. The HCV genome is a positive-strand RNA of 9.6 kb encoding a polyprotein that is post-translationally processed into structural (Core, E1, E2 and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A and NS5B) proteins. In the present work, a variety of computational methods and approaches are applied to investigate HCV proteins involved both in the fusion process mechanism (E1 and E2 envelope glycoproteins) and drug resistance (NS3 protease). The E1/E2 glycoprotein complex represents the surface of the virus which is largely responsible for virus antigenicity and is involved in important viral processes including virus attachment and cell-entry. The NS3/4A protease is responsible for several important biological functions in the HCV life cycle, including polyprotein cleavage, viral replication and inhibition of the host antiviral response. The protease domain is one of the main candidate targets for rational drug design. Today, structural knowledge about both the E1 and E2 glycoproteins is very limited. This is due to the lack of feasible expression systems for etherologous proteins, which has made attempts at crystallization unsuccessful. However, two models of the three-dimensional structure of E2, obtained by fold recognition have been proposed by Yagnik et al. in 2000 (Model-1) and by Spiga et al. in 2006 (Model-2). In both cases, the molecular model is based on the E protein of Tick-Borne Encephalitis Virus (TBEV), a virus belonging to the Flaviviridae family and thus evolutionary closely related to HCV. These models are compared and evaluated in terms of their reliability according to experimentally derived functional information. Model-1 seems to be the most consistent with the functions of E2 as supported by collected evidence. However, this model presents some weak points, the most noteworthy being that is does not take into account the location of the strictly conserved cysteine-residues forming nine disulphide bonds. However, the recently acquired knowledge of the E2 protein disulphide bonds, and experimentally derived findings provide sufficient constrains to reconstitute a new model not only for this protein but also for the E1/E2 complex. The current E1E2 model is constructed using the E1 glycoprotein of alphavirus Semliki Forest Virus (SFV) a Class II fusion protein as template. Class II fusion proteins are elongated molecules composed almost entirely of β-strands containing three domains. Domain I is connected to domain II by a highly flexible hinge region. The fusion loop is located on the tip of the domain II, loop crucial for the fusion mechanism. The immuno globulin-like domain III is located in a lateral position and is followed by a stem region connecting the protein ectodomain to the its transmembrane domain. Our HCV E2 model matches well with domains I and II of the template fusion protein while HCV E1 matches domain III. The most important feature of our model is that it takes into account the location of the strictly conserved cysteine residues forming nine disulphide bonds. Validation of this model is performed mapping the most important functional sites. The localization of principal functional sites is often in agreement with experimental data obtained so far. Furthermore there are some proposed novel features of HCV envelope proteins which present a structural hypothesis explaining the viral membrane fusion machinery architecture. This new E1E2 model shares the main features of Class II fusion proteins. In this case the fusion process is promoted by a dimer of two proteins instead of the single one in Class II proteins. The proposed E1E2 fusion complex involves E1 as anchor for the entire structure, and makes contact with E2 by their respective transmembrane and stem regions. An α-helix insertion in E1 also increases the interaction surface between them. In E2, the flexible loop together with the hinge region has a principal role in conformational changes during the fusion process. Moreover, the new model places the fusion loop very well on the tip of the elongated domain II, in which the GWG motif (mostly conserved among E2 HCV genotypes and the other members of the same family) is very exposed. This is important because we think that it is the principal structural feature of this sequence stretch of directly involved in the insertion into the host cell membrane, and it is able to bridge the gap between the viral and cell membranes to promoting their fusion. In the last part of this thesis the attention has been focused on the use an emergent method: residue interaction networks (RINs) where each node represents a residue in the protein and connections are used to indicate different interaction types (van-der-Waals contacts, salt bridges, pi-pi stacks or simple hydrophobic contacts). We used residue interaction networks to investigate molecular effects underlying drug resistance in the NS3 protease, one the current candidate target to develop inhibitors. All published NS3data about both natural occurring variants and drug induced mutations associated to a decrease susceptibility to protease inhibitors were collected. Attention has been focused on the study of drug resistance mechanisms against the two inhibitors Telaprevir and Boceprevir, currently in phase III of clinical trial. Two variants V36M and R155K, have been analyzed. The V36M variant affects the local conformation and the geometry of the hydrophobic cavity, as a consequence of the a higher number of interactions which confers higher rigidity to this site if compared with the WT strain. The mutations effect is reflected on the immediately close active site binding pocket. The R155K variant has an impact both on the local conformation in proximity of the beta-barrel domain involved in substrate binding but also in the active site binding pocket. In this case RIN analysis showed the importance of G140 located in the same loop as S139 (catalytic triad amino acid directly involved in inhibitors binding). G140 probably plays an important role in maintaining the flexibility of this loop in the WT strain while in the mutated protein this condition is lost due to an increased number of interactions such as a a new hydrogen bond with an amino acid responsible for substrate binding (F154) and directly interacting with S139. Applying filters based on residue conservation and their degree (number of interactions per residue), it has been possible to identify functionally and structurally important residues. As expected, some of these are part of functionally important sites such as the catalytic triad, hydrophobic cavity and substrate binding sites. Other are not involved in the known NS3function but probably, on the basis of these results, are critical to maintain the NS3structure.
Nel mondo circa 200-300 milioni di persone sono cronicamente infettate dal virus dell’Epatite C (HCV). Nel 20% dei casi la cronicità può portare a cirrosi ed epatocarcinoma. HCV fa parte della famiglia dei Flaviviridae, come Dengue Virus e West Nile Virus (WNV), ed è classificato nel genere Hepacivirus, per le differenze nella sequenza amminoacidica. Il genoma di HCV è costituito da un singolo filamento di RNA a polarità positiva di 9.6 kb che codifica per un'unica poliproteina la quale viene successivamente processata nelle rispettive proteine strutturali (Core, E1, E2, e p7) e nelle proteine non strutturali (NS2, NS3, NS4, NS4B, e NS5B). In questa tesi, sono stati applicati una serie di metodi computazionali e differenti approcci per studiare proteine del virus dell’ epatite C (HCV) coinvolte nel processo di fusione (le glicoproteine proteine dell’envelope E1 e E2) e nel meccanismo di resistenza ai farmaci. Le glicoproteine E1 e E2, costituiscono la superficie del virus e sono responsabili delle sue proprietà antigeniche. Sono inoltre coinvolte nel processo di interazione con la membrana della cellula ospite e dell’entrata del virus al suo interno. La proteina NS3/4A (proteasi virale) è responsabile di una serie di importanti funzioni nel ciclo di replicazione virale che includono: il processamento della poliproteina nelle rispettive proteine strutturali e non strutturali, la replicazione del virus e inibizione della risposta antivirale della cellula ospite. La proteasi è uno dei candidati target per la progettazione di farmaci antivirali. Al momento, la conoscenza delle caratteristiche strutturali delle glicoproteine E1 e E2 è molto limitata. Ciò è dovuto dalla mancanza di un sistema eterologo di espressione di queste proteine che rende difficoltosa cristallizzazione. Nonostante la mancanza di una struttura cristallografica, sono stati proposti due modelli tridimensionali per la proteina E2. Questi modelli sono stati ottenuti col metodo bioinformatico del “fold recognition” e sono stati proposti dal gruppo di Yagnik nel 2000 (Modello-1) e dal gruppo di Spiga nel 2006 (Modello-2). In entrambi i casi, il modello si basa sulla glicoproteina E dell’envelope del virus Tick-Borne Encephalitis virus (TBEV), un virus appartenente alla famiglia Flaviviridae e quindi evolutivamente correlata all’HCV. Questi due modelli sono stati comparati e valutati per la affidabilità considerando le informazioni ottenute sperimentalmente. Sulla base di questi risultati, il Modello-1 sembra essere più coerente con le funzioni di E2 come supportato dalle evidenze sperimentali. Tuttavia questo modello presenta dei punti deboli, il più importante è il fatto che non tiene conto del pattern delle cisteine che formano nove ponti disolfuro. La recente identificazione delle cisteine formanti i ponti disolfuro, e le evidenze sperimentali, hanno fornito una base sufficiente per costruire un nuovo modello, non solo della proteina E2 ma del complesso E1E2. L’attuale modello è stato costruito usando la glicoproteina E1 del virus Semliki Forest Virus (SFV) come templato, un virus appartenente al genere alphavirus e alla famiglia Togaviridae. La glicoproteina E1 di SVF appartiene alle proteine si fusione virali di classe II che sono strutture allungate composte quasi interamente da foglietti β e contengono tre domini. Il dominio I è connesso al dominio II tramite una regione cerniera molto flessibile (Hinge region). All’estremità del dominio II è localizzato il Il loop di fusione che ha un ruolo fondamentale nel processo di fusione. L’ immuno globuline-like dominio III, è localizzato lateralmente ed è seguito da una regione detta “stem region” che connette il dominio esterno al dominio transmenbrana. Il nostro modello della proteina E2 di HCV si adatta molto bene col dominio I e II, mentre E1 si adatta col dominio III della proteina di fusione usata come templato ed è in accordo col il pattern delle cisteine che formano i ponti disolfuro. Inoltre la bontà della struttura risultante è stata valutata mappando i siti funzionali più importanti e, la loro localizzazione è spesso in accordo con i dati sperimentali. Il modello E1E2 ha inoltre permesso di proporre una nuova ipotesi che spiega il meccanismo della fusione del virus con la membrana della cellula ospite. Questo modello inoltre condivide con le proteine di fusione di classe II una serie di caratteristiche, tranne il fatto che in queste ultime, la proteina di fusione, e il processo di fusione è promosso da un'unica proteina (E1) mentre in HCV da due proteine, E1 e E2. Il complesso di fusione E1E2 presentato in questo lavoro, propone l’ipotesi che E1 possa fungere da ancoraggio per l’intera struttura e inoltre prendere contatti con la proteina E2 mediante le rispettive regioni trans membrana e regioni “stem”. La presenza di un α elica nel modello di E1 incrementa l’interazione tra le due proteine. In E2, il loop flessibile situato nella regione “stem”, insieme alla regione cerniera “hinge”, svolge un ruolo principale durante i cambiamenti conformazionali a cui sono sottoposte queste proteine durante il processo di fusione. Nel modello è localizzato correttamente il loop di fusione nel quale, il motivo GWG (molto conservato nelle sequenze di E2 dei diversi genotipi) è molto esposto. Questo è molto importante perché pensiamo che il motivo GWG sia la più importante caratteristica strutturale/funzionale presente nel loop di fusione che lo vede direttamente coinvolto nell’inserzione nella membrana cellulare ospite e capace quindi di colmare il divario tra la membrana cellulare della cellula ospite e del virus, promuovendo la loro fusione. Nell’ultima parte del lavoro, l’attenzione è stata focalizzata sull’uso di un metodo emergente “reti di interazione dei residui amminoacidici” (RINs) dove ogni nodo corrisponde ad un amminoacidico della proteina e le connessioni rappresentano i diversi tipi di interazione (contatti van-der-Waals, ponti salini, legami π-π o semplici contatti idrofobici). Le reti di interazioni sono state utilizzate per studiare l’effetto molecolare che determina la resistenza ai farmaci nella proteina NS3 (proteasi di HCV), uno dei target per lo sviluppo di inibitori. Per questo studio sono state collezionate tutte le mutazioni associate alla resistenza indotta da due farmaci, Telaprevir e Boceprevir, attualmente in fase III di sperimentazione clinica e sono state analizzate due varianti V36M e R155K. La variante V36M influisce sulla conformazione locale e sulla geometria della cavità idrofobica della proteina, questo effetto è una conseguenza del fatto che la mutazione stabilisce un maggior numero di interazioni nella proteina mutata rispetto alla proteina WT. Questo effetto si riflette anche sulla tasca del sito attivo localizzato vicino ad essa. Nella variante R155K, invece l’effetto della mutazione si riflette sul cambiamento conformazionale in corrispondenza del domino ß-barrel coinvolto nel binding con il substrato e di conseguenza sulla vicina tasca del sito attivo. In quest’ultima analisi, le reti di interazione amminoacidiche hanno evidenziato l’importanza del residuo G140, localizzato nello stesso loop del residuo S139 (amminoacido del sito catalitico anche direttamente coinvolto nel legame con gli inibitori). G140 probabilmente ha un ruolo fondamentale nel mantenimento della flessibilità di questo loop. Nella proteina mutata questa flessibilità viene persa in conseguenza al fatto che G140 ha un maggior numero di interazioni, in particolare un nuovo legame idrogeno con l’amminoacido F154, direttamente coinvolto nel legame col substrato. Il residuo F154 interagisce direttamente con S139. Applicando filtri basati sulla conservazione e sul grado dei nodi (totale numero di interazioni di ogni residuo nella rete), è stato possibile identificare residui importanti sia dal punto di vista funzionale che strutturale. Come ci si aspettava, alcuni residui non sono conosciuti come funzionalmente importanti, ma questi probabilmente, sulla base dei risultati ottenuti potrebbero essere critici per il mantenimento della conformazione strutturale.

O desenvolvimento de uma vacina para o controle da dengue é uma prioridade em todo o mundo. O domínio helicase da proteína NS3 (NS3H) viral alberga epítopos reconhecidos por linfócitos T citotóxicos, os quais tem papel importante na eliminação de células infectadas. Esse trabalho propôs a obtenção de uma forma recombinante, produzida em linhagens de Escherichia coli, da NS3H do DENV2 com características similares à proteína nativa e sua utilização como um potencial antígeno vacinal. A NS3H foi obtida na forma solúvel, foi reconhecida por anticorpos de camundongos e de humanos infectados e foi capaz de interagir com o RNA viral. Camundongos imunizados com NS3H coadministrada com diferentes adjuvantes desenvolveram respostas imunológicas específicas mas não foram protegidos após desafio. Em conjunto, os resultados indicam que a proteína NS3H recombinante preserva conformação e determinantes antigênicos da proteína viral nativa e pode ser útil em estudos sobre a biologia viral e na busca de estratégias anti-virais voltadas para o controle da dengue.
The development of a dengue vaccine is a worldwide priority. The helicase domain of viral NS3 protein (NS3H) preserves epitopes recognized by cytotoxic T lymphocytes, which plays an important role in the elimination of infected cells. This study aimed the generation of a recombinant NS3H form of a type 2 dengue virus (DENV2) lineage, in Escherichia coli strains, with properties similar to the native protein and its use as a potential vaccine antigen. The NS3H was obtained in soluble form, was recognized by antibodies from mice and human subjects and was able to interact with the viral RNA. Mice immunized with NS3H combined with different adjuvants developed specific immune responses but did not confer protection to a lethal challenge. Altogether, the results indicate that the recombinant NS3H protein preserves conformational and antigenic determinants of the native protein and may be a useful tool for studies dealing with the DENV biology and the search for anti-virus approaches.

La tesi si incentra sullo studio del simulatore Network Simulator 3 per la simulazione di reti LTE. Si inizia con l'introduzione allo standard LTE, definendone le tecnologie utilizzate e le innovazioni apportate alle reti di comunicazione. Si introduce, quindi, il simulatore ns-3 e ci si sofferma sulle funzionalità utilizzate. Si prosegue con la presentazione e la valutazione di simulazioni del sistema LTE in scenari con uno o due eNodeB e uno o due utenti mobili per verificare il funzionamento del simulatore e del sistema LTE in termini di throughput, ritardi e di funzionalità di handover. Infine, si presentano simulazioni della rete LTE nello scenario di Bologna tramite l'utilizzo del route mobility model per la generazione di traffico realistico. I risultati mostrano il throughput medio percepito da ciascun utente in funzione della copertura effettuata e suggeriscono potenziali sviluppi futuri in termini di copertura e sviluppo di rete.

Innovative distributed systems are often studied with the aid of simulation, especially in the case of large scale and situated systems. One of the key aspects of distributed systems is the presence of a set of nodes which must communicate with each other in order to perform their collective task. Consequently, the behaviour of the network plays a key role in determining how the distributed system will act as a whole, but support for realistic simulation of network communication may not be available in simulators that focus on higher-level phenomena, such as the execution of a program on the nodes belonging to a distributed system. Network simulation is usually performed with dedicated simulators which, on the other hand, mostly focus on low-level aspects, such as the behaviour of the physical channels and of the network protocols. The present works aims at filling this gap between high-level distributed system simulation and low-level network simulation by creating a cross-simulator integration between Alchemist, a simulator for large scale situated distributed systems, and ns3, a network simulator, which has been exploited in order to give Alchemist the ability to accurately simulate the network interactions between the nodes. Finally, the whole system has been tested to demonstrate how different network setups can affect the execution of a program in a distributed system.

West Nile virus (WNV) is the most widely distributed arthropod-borne virus globally. It can cause a potentially fatal infection and has become a public health concern in North America since its introduction in 1999. Currently, there are no vaccines or treatments available for human WNV infections. As such, it is important to understand the virus life cycle, in order to develop effective therapeutics. The WNV protease heterocomplex, NS2B/NS3, is a prime target for antiviral therapy and has become the focus of much research. It is important to understand protease function first, in order to develop effective inhibitors. The overall goal of this thesis was to gain a better understanding into the function of the full-length NS2B/NS3 protease heterocomplex within the intracellular microenvironment. I hypothesized that there are critical residues essential for the interaction between NS2B and NS3 that affect protease activity and protein stability. The first aim of this project was to generate a cell-based fluorescent substrate assay to investigate the protease activity of the full-length NS2B/NS3 protease heterocomplex within the cell. My results demonstrate that the full-length NS2B/NS3 protease heterocomplex functions differently within the context of the cell, compared to what has been previously observed in vitro (Chapter 2). In the second aim, I investigated NS2B function on NS3 protease cis-cleavage and trans-cleavage activity. My results reveal an important dual role the NS2B protein plays in the proper function of the full-length NS2B/NS3 protease heterocomplex (Chapter 3). In the third aim, I utilized the information gathered to rationally design and test a serine protease inhibitor directed against the full-length NS2B/NS3 protease heterocomplex (Chapter 4). Taken together, my results highlight the importance of utilizing cell-based assays to assess protease activity, as this allows for the investigation of NS2B/NS3 protease function in a more physiologically relevant environment. The results presented in this thesis further our understanding of the activity of the full-length WNV NS2B/NS3 protease heterocomplex within the context of the cell. The information gathered gives insight into the regulation of viral protease function that could be utilized in the rational drug design towards the WNV NS2B/NS3 protease heterocomplex.

HSPA planning requires identification of key performance indicators  (KPIs), such as infrastructure cost,performance, service quality and the amount of E/M pollution. To evaluate such a framework describing and linking these key elements for HSPA planning and optimization we need to carry out a set of HSPA system-level event-driven simulations under different scenarios. To have such simulations, a new tool should be developed. In this thesis work a simulation tool is developed within NS-3, an open source network simulator. The simulation tool consists several entities to represent a UMTS network and respective nodes in it, and necessary procedures to form a network dynamically. A simple base station removal algorithm in a system level, is developed focusing on the reduction of capital expenditure (CAPEX) of a network. The algorithm defines a cost function in order to remove the most costly base station from network preserving the target coverage percentage. Baseline results show that the simulation tool is working properly and later the results from algorithm indicate that, it is possible to use the  algorithm within a dynamic simulation.

The Hepatitis C virus (HCV) NS3/4A protease inhibitors (PIs) have become a mainstay of newer all-oral combination therapies. Despite improvements in potency of this inhibitor class, drug resistance remains a problem with the rapid emergence of resistance-associated substitutions (RASs). In this thesis I elucidate the molecular mechanisms of drug resistance for PIs against a resistant variant and apply insights toward the design of inhibitors with improved resistance profiles using structural, biochemical and computational techniques. Newer generation PIs retain high potency against most single substitutions in the protease active site by stacking on the catalytic triad. I investigated the molecular mechanisms of resistance against the Y56H/D168A variant. My analysis revealed that the Y56H substitution disrupts these inhibitors’ favorable stacking interactions with the catalytic residue His57. To further address the impact of drug resistance, I designed new inhibitors that minimize contact with known drug resistance residues that are unessential in substrate recognition. The initially designed inhibitors exhibited flatter resistance profiles than the newer generation PIs but lost potency against the D168A variant. Finally, I designed inhibitors to extend into the substrate envelope (SE) and successfully regained potency against RAS variants maintaining a flat profile. These inhibitors both pack well in the enzyme and fit within the SE. Together these studies elucidate the molecular mechanisms of PI resistance and highlight the importance of substrate recognition in inhibitor design. The insights from this thesis provide strategies toward the development of diverse NS3/4A PIs that may one day lead to the eradication of HCV.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A proteína NS3 apresenta dois domínios e é bifuncional. Apresenta três funções enzimáticas que são; 1) atividade de protease; 2) NTPase e 3) helicase. A função protease relaciona-se a tradução da proteína precursora e as funções NTPase e helicase tem grande participação na replicação do material genético viral. Trata-se de uma molécula essencial para o processamento da poliproteína precursora e também para a replicação viral e portanto, um dos principais alvos para o desenvolvimento de drogas antivirais. No domínio Protease foram evidenciadas substituições na tríade catalítica e na região de ligação ao íon zinco nos pacientes avaliados. Estas substituições, quando somadas podem explicar a resposta ao tratamento. Também foram visualizadas alterações na porção Helicase da NS3. As substituições ocorreram nos sítios de ligação ao ATP e ao RNA. Outros resíduos da Helicase relevantes para o desenvolvimento de inibidores, como R2133 e F258 e F264 não apresentaram substituições, evidenciando tratarem-se de aminoácidos conservados nessa região. Os resultados obtidos nesse trabalho fornecem informações sobre o perfil genético do vírus HCV do genótipo 3 especificamente da região codificadora da proteína NS3, permitindo o conhecimento do genoma viral e a identificação de regiões para ligação de possíveis inibidores. Este projeto certifica que a modelagem é uma ferramenta útil para a biologia estrutural e funcional, e que os modelos obtidos aqui contribuem para o desenho de novas drogas anti-virais específicas para o genótipo 3 do vírus HCV
The NS3 protein has two domains and is bifuntional. It presents three functions: 1) protease activity, 2) NTPase and 3) helicase. The protease function is related to the translation of the poliprotein precursor and functions NTPase and helicase has great participation in the replication of the viral genetic material. So. The NS3 is considered the major target for the development of antiviral drugs. In the Protease portion substitutions were evidenced in catalytic triad and the zinc ion binding sites, in the patients evaluated. These substitutions, when added up can explain the response to treatment. Also were observed changes in Helicase portion of NS3. The substitutions took place on ATP and RNA binding sites. Other residues of Helicase relevant to the development of inhibitors, as R2133 and F258 and F264, showed no substitutions, highlighting the great conservation of amino acids in this region. The results obtained in this work provide information on the genetic profile of the HCV virus genotype 3, specifically the region of NS3 protein, allowing the knowledge of the viral genome and the identification of regions for possible connection of inhibitors. This project certifies that the modeling is a useful tool for structural biology and functional, and that the models obtained here contribute to the design of new anti-viral drugs specific to the genotype 3 of HCV virus

O crescimento dos serviços de banda-larga em redes de comunicações móveis tem provocado uma demanda por dados cada vez mais rápidos e de qualidade. A tecnologia de redes móveis chamada LTE (Long Term Evolution) ou quarta geração (4G) surgiu com o objetivo de atender esta demanda por acesso sem fio a serviços, como acesso à Internet, jogos online, VoIP e vídeo conferência. O LTE faz parte das especificações do 3GPP releases 8 e 9, operando numa rede totalmente IP, provendo taxas de transmissão superiores a 100 Mbps (DL), 50 Mbps (UL), baixa latência (10 ms) e compatibilidade com as versões anteriores de redes móveis, 2G (GSM/EDGE) e 3G (UMTS/HSPA). O protocolo TCP desenvolvido para operar em redes cabeadas, apresenta baixo desempenho sobre canais sem fio, como redes móveis celulares, devido principalmente às características de desvanecimento seletivo, sombreamento e às altas taxas de erros provenientes da interface aérea. Como todas as perdas são interpretadas como causadas por congestionamento, o desempenho do protocolo é ruim. O objetivo desta dissertação é avaliar o desempenho de vários tipos de protocolo TCP através de simulações, sob a influência de interferência nos canais entre o terminal móvel (UE User Equipment) e um servidor remoto. Para isto utilizou-se o software NS3 (Network Simulator versão 3) e os protocolos TCP Westwood Plus, New Reno, Reno e Tahoe. Os resultados obtidos nos testes mostram que o protocolo TCP Westwood Plus possui um desempenho melhor que os outros. Os protocolos TCP New Reno e Reno tiveram desempenho muito semelhante devido ao modelo de interferência utilizada ter uma distribuição uniforme e, com isso, a possibilidade de perdas de bits consecutivos é baixa em uma mesma janela de transmissão. O TCP Tahoe, como era de se esperar, apresentou o pior desempenho dentre todos, pois o mesmo não possui o mecanismo de fast recovery e sua janela de congestionamento volta sempre para um segmento após o timeout. Observou-se ainda que o atraso tem grande importância no desempenho dos protocolos TCP, mas até do que a largura de banda dos links de acesso e de backbone, uma vez que, no cenário testado, o gargalo estava presente na interface aérea. As simulações com erros na interface aérea, introduzido com o script de fading (desvanecimento) do NS3, mostraram que o modo RLC AM (com reconhecimento) tem um desempenho melhor para aplicações de transferência de arquivos em ambientes ruidosos do que o modo RLC UM sem reconhecimento.
The growth of broadband services in mobile networks has led to a demand for data with faster and better quality transmissions. The mobile network technology called LTE (Long Term Evolution) or fourth generation (4G) came up with the objective of attending this demand for wireless access to services such as Internet access, online games, VoIP and video conferencing. LTE is part of the specifications of 3GPP Releases 8 and 9 operating in all-IP networks and providing transmission rates above 100 Mbps (DL), 50 Mbps (UL), low latency (10 ms) and compatibility with previous versions of mobile networks, 2G (GSM / EDGE) and 3G (UMTS / HSPA). The TCP protocol designed to operate in wired networks presents poor performance over wireless channels such as mobile cellular networks, due mainly to the characteristics of selective fading, shadowing and high error rates coming from the air interface. As all losses are interpreted as caused by congestion the protocol performance is bad. The objective of this dissertation is to evaluate the performance of several types of the TCP protocols through simulations, under the influence of channel interference between the mobile terminal (UE - User Equipment) and a remote server. For this, the NS3 (Network Simulator version 3) software and the protocols TCP Westwood Plus, New Reno, Reno and Tahoe were used. Results have shown that the TCP Westwood Plus protocol has a better performance than others. The New Reno and Reno TCP protocols had similar performance due to the proposed interference model, which has a uniform distribution and so the possibility of loss of consecutive bits is low on the same transmission window. TCP Tahoe, as expected has shown the worst performance among all because it does not have the fast recovery mechanism and its congestion window keeps coming back to one segment after a timeout. It was also observed that the delay has a greater importance in the performance of TCP when comparing with the bandwidth of the access and backbone links importance, once in the tested scenario the bottleneck was present in the air interface. The simulation performed with noise in the Air Interface, introduced by the NS3 fading script, showed that the RLC AM (acknowledged mode) had a better performance than the RLM UM (Unacknowledged mode).

Factors that determine the molecular basis of African horsesickness virus (AHSV) virulence are unclear. Several proteins and different events in the viral replication cycle together with different environmental factors are likely to contribute to the virulence phenotype. The aim of this investigation was to study the possible contribution of AHSV nonstructural protein NS3 to virus virulence. NS3 is a cytotoxic protein that localizes in areas of plasma membrane disruption and is assumed to be associated with the release of virus particles from infected cells. A conserved feature in all AHSV NS3 proteins is the presence of two hydrophobic domains. To investigate whether these hydrophobic domains interact with membranes, cell surface localization and membrane association studies were conducted on NS3 and the two hydrophobic domain mutant forms of NS3. Results indicated that mutations in either hydrophobic domain did not prevent membrane targeting but abolished membrane anchoring. This prevented cell surface localization and obviated the cytotoxic effect of NS3. AHSV NS3 is a highly variable protein. This level of variation may influence virulence properties. To compare the NS3 sequence variation level of South African AHSVs and bluetongue viruses (BTV/s) with those previously described, the NS3 protein sequences of field isolates, reference and vaccine strains were determined and analysed. The variation level of AHSV NS3 was found to be higher than previously reported. Comparison of the AHSV vaccine and field strain NS3 sequences revealed no obvious NS3 virulence marker. The level of AHSV NS3 sequence variation could distinguish between field isolates and vaccine strains and differentiate between sub-populations within a serotype. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters with exception of AHSV-8 and one AHSV-6 encoded NS3. BTV NS3 inferred phylogeny indicated that the three BTV NS3 clusters that occur as geographically defined entities are all simultaneously present in S.A. This would suggest that BTV originated in southern Africa. To investigate the impact of minor NS3 variation on AHSV virulence, the membrane permeabilization properties of AHSV-2 vaccine and virulent NS3 proteins were compared. The AHSV-2 S10 genes were cloned and NS3 was expressed using the BAC-TO-BAC system. The membrane permeabilization effect of NS3 expression was monitored by calcium influx into insect cells. Expression of either the vaccine or virulent associated AHSV-2 NS3 protein equally increased membrane permeability. The NS3 sequence variation therefore had a limited influence on membrane permeabilization properties and the virulence status of the vaccine and virulent AHSV-2 strains in this study. The possible effect of larger NS3 sequence variation levels on AHSV virulence was investigated by NS3 associated properties such as virus release and membrane permeabilization. AHSV infections increased the permeability of cell membranes and the different strains had different virus release properties. Virus release was not exclusively related to increased membrane permeability of infected cells or to the yield of the virus in the infected cells. The level of NS3 variation may influence AHSV release mechanisms and membrane permeabilization properties thereby contributing to AHSV virulence properties.
Thesis (PhD (Genetics))--University of Pretoria, 2005.
Genetics
unrestricted

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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
Dengue infection has become a public health problem in tropical and sub-tropical areas. It is a viral disease transmitted by arthropods with high mortality rates. Its clinical diagnosis requires laboratory confirmation because several other conditions can also cause similar symptoms with acute fevers as well as the primary use antibodies or antigens for detection of DENV. The continued development of diagnostic tests that are cheap, sensitive, specific, easy to perform, and that are capable of giving early diagnosis of the dengue virus infection is still a needed. Using bioinformatics, we mapped peptides of Envelope protein (E), Nonstructural 1 and 2 which have antiginity, hidrofility and probality to apper on the surface. The first peptide, with molecular weight of the 17KDa, was called E/ENS3 and corresponds to two Envelope protein (E) sequences and a Nonstructural 3 sequence. The second peptide, with molecular weight of the 15Kda, was called ENS1/3 and it corresponds to a Envelope protein (E) sequence, a Nonstructural 1 sequence and Nonstructural 3 sequence. The peptides were expressed in prokaryotic sistem, purified and used in ELISA (Enzyme-Linked Assay Imunoabsorvente). Using Indirect ELISA in the IgM and IgG antibody detection previously characterized, we obtained as results: (1) IgG research using E/ENS3 and ENS1/3 peptides, sensibility was 65% and the specificity was 91,7% and (2) IgM research, sensibility was 75,7% and and the specificity was 91,8%. These results indicate that use of both E/ENS3 and ENS1/3 peptides may be a alternative to the serological diagnosis of dengue.
A infecção pelo vírus dengue continua sendo um grande problema de saúde pública em regiões tropicais e subtropicais do mundo, sendo a principal doença viral transmitida por artrópodes, com alta morbidade e mortalidade. O diagnóstico clínico da dengue requer confirmação laboratorial devido à semelhança dos sintomas com uma série de outras febres agudas e para detecção de infecções virais primárias ou secundárias. O desenvolvimento de testes baratos, sensíveis, específicos e de fácil execução, que sejam capazes de proporcionar diagnóstico precoce da infecção pelo vírus da dengue, é ainda uma necessidade. Neste trabalho mapeamos, por ferramentas de bioinformática, peptídeos das proteínas de Envelope (E), Não Estrutural 1 e 2 que apresentasse como características: elevada antigenicidade, hidrofilicidade e probabilidade de se apresentar na superfície. O primeiro peptídeo, com peso molecular de 17KDa, foi denominado E/ENS3 e corresponde a duas sequências da proteína de Envelope (E) e uma sequência da Não Estrutural 3. O segundo peptídeo, com peso molecular de 15Kda, foi denominado ENS1/3 e corresponde a uma sequência da proteína de Envelope (E), uma sequência da Não Estrutural 1 e uma sequência da Não Estrutural 3. Os peptídeos foram expressos em sistemas em sistema procarioto, purificados e utilizados em teste de ELISA (Enzyme-Linked Assay Imunoabsorvente). Foram realizadas a detecção de anticorpos IgM e IgG de amostras de laboratório, previamente caracterizadas, através de ELISA Indireto e obteve-se os seguintes resultados: (1) na pesquisa de IgG usando os peptídeos E/ENS3 e ENS1/3, a sensibilidade foi de 65% e a especificidade foi de 91,7% e (2) na pesquisa de IgM a sensibilidade foi de 75,7% e especificidade foi de 91,8%. Estes resultados indicam que a utilização dos peptídeos E/ENS3 e ENS1/3 podem ser uma alternativa para o diagnóstico sorológico da dengue

Hepatitis C virus (HCV) is a major cause of viral hepatitis. The World Health Organization (WHO) estimates the global prevalence of HCV to be approximately 2.2-3.0% with 130-170 million infected individuals worldwide. HCV has the ability to evade the immune system and to establish persistent infections, which often result in chronic liver disease. HCV encodes a long polyprotein which contain structural and non-structural proteins. The Non-structural protein NS3 is likely to be involved in viral RNA replication by interacting with viral and host components. In this study, a purified recombinant full-length NS3 protein expressed in E. coli was examined for its ability to interact with biotinylated full length HCV JFH-1 genomic RNA and the 3' terminal of HCV negative strand RNA. The results showed that NS3 could bind to both full length and the 3' end negative strand RNAs. RNA interference (siRNA) studies were also carried out to investigate the role of host genes RAB40B, RAB27B, TXNIP and Staufen 1, which had previously been shown to be important for HCV replication, on NS3 and NS3/4A proteins expressed by adenovirus vectors using quantitative real-time polymerase chain reaction (qRT-PCR) and FACS analysis. While there were reductions in the levels of the NS3 and NS3/4A transcripts when the expression of these host genes was knocked down by siRNAs, the NS3 and NS3/4A proteins appeared to be more stable when RAB40B, RAB27B, but not TXNIP or Staufen 1, were knocked. One possibility is that although silencing of these host genes may reduce HCV replication, this may also increase in the stability of NS3 or NS3/4A proteins by altering their location within the cell due to the absence of these host proteins. Previously it has been shown that NS3 and NS3/4A protein expression decreased the level of expression of several proteins, which were part of the innate immune response, leading to interferon production. In this study, it was found that the expression of NS3 and NS3/4A proteins, using adenovirus vectors, restored RIG-I, MDA5 and MAVS expression levels in response to dsRNA stimulation, indicating that the NS3 protein may have a role in regulating the expression of these host genes.

Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.

Dengue virus (DENV) is a serious worldwide health concern putting about 2.5 billion people in more than 100 countries at-risk Dengue is a member of the flaviviridae family, is transmitted to human via mosquitos. Dengue is a deadly viral disease. Unfortunately, there are no vaccines or antiviral that can prevent this infection and that is why researchers are diligently working to find cures. The DENV genome codes for multiple nonstructural proteins one of which is the NS3 enzyme that participates in different steps of the viral life cycle including viral replication, viral RNA genome synthesis and host immune mechanism. Recent studies suggest the role of fatty acid biogenesis during DENV infection, including posttranslational protein modification. Phosphorylation is among the protein post translational modifications and plays essential roles in protein folding, interactions, signal transduction, survival and apoptosis.

In silico study provides a powerful approach to gain a better understanding of the biological systems at the gene level. NS3 has the potential to be phosphorylated by any of the ∼500 human kinases. We predicted potential kinases that might phosphorylate NS3 and calculated Dena ranking score using neural network and other machine learning based webserver programs. These scores enabled us to investigate and identify the top kinases that phosphorylate DENV NS3. We hypothesize that preventing the phosphorylation of NS3 may interrupt the viral replication and participate in antiviral evasion. Using multiple sequence alignment bioinformatics tools we verified the results of the highly conserved residues and the residues around active sites whose phosphorylation may have a potential effect on viral replication. We further verified the results with multiple bioinformatics tools. Moreover, we included the Zika virus in our research and analysis taking into consideration the facts that Zika is related to the dengue virus because it belongs to the same Flavivirus genus affecting humans which might lead to a lot of similarities between Zika and Dengue, and that Zika is available for in vitro testing.

Our studies propose that the Host-Mediated Phosphorylation of NS3 would affect its capability to interact with NS5 and knocking out one of the interacting proteins may inhibit viral replication. These results will open new doors for further investigation and future work is expected to help identify the key inhibition mechanisms.

African horsesickness virus (AHSV) is a double-stranded RNA virus belonging to the Orbivirus genus in the Reoviridae family (Bremer et al., 1990; Calisher and Mertens, 1998). The virus is highly pathogenic and its mortality rate in horses, the most susceptible species, may be as high as 95% (House, 1993). S10, the smallest genome segment of AHSV, codes for two proteins (NS3 and NS3A) from in-phase overlapping reading frames. The C-terminal sequences of these proteins are identical, but NS3A lacks the first 10 amino acids present on the N-terminal of NS3 (Van Staden and Huismans, 1991). Nonstructural protein NS3 is a membrane protein, associated with both smooth intracellular membranes and the plasma membrane. NS3 has pleiotropic roles in the viral life cycle including the transport and release of mature virions and viroporin-like alteration of cell membrane permeability. NS3 is cytotoxic when expressed in bacterial or insect cells, and is speculated to play a vital role in viral virulence and disease pathogenesis (Stoltz et al., 1996; Van Staden et al., 1995). A number of different domains that could mediate the membrane interaction or intracellular trafficking of NS3 have been identified. The relative contributions of these domains in insect and mammalian cells are not known, but could differ, as there are distinct differences in NS3 expression levels, cytopathic effects and virus release mechanisms in these two cell types. In order to investigate the subcellular localisation of NS3, a number of full-length, truncated or mutant versions of AHSV-3 NS3 were constructed as C-terminal eGFP (enhanced green fluorescent protein) fusion proteins. These proteins were used to generate recombinant baculoviruses for expression in Spodoptera frugiperda (Sf9) insect cells and were compared in terms of their subcellular localisation by conventional fluorescence microscopy. Confocal laser microscopy was used to investigate co-localisation with the nucleus, the Golgi apparatus and the Endoplasmic Reticulum (ER). Subcellular fractionations and membrane flotation analyses were used to confirm membrane interactions and to identify detergent-resistant membrane fractions. NS3 as well as a C-terminal deletion of NS3 targeting a putative dileucine motif both localised to cellular/nuclear membrane components. In contrast, site-specific mutations to either of the transmembrane domains abolished membrane association and resulted in cytoplasmic localisation. NS3A showed mixed results, displaying both membrane localisation and a cytoplasmic distribution. The 11 amino acid region unique to NS3 and absent from NS3A, which has been shown to bind to cellular exocytosis proteins in bluetongue virus (Beaton et al., 2002), did not display membrane interaction. These results indicate that both of the hydrophobic domains as well as the N11 region are required to be present for NS3 to be properly targeted to the plasma/nuclear membrane. In addition, NS3 was shown to be present in detergent-resistant membrane fractions, indicative of a possible localisation within lipid rafts. The above results indicate that the NS3 protein contains specific signals involved in membrane targeting, confirming a potential role for NS3 in viral localisation and release in the AHSV replication cycle.
Dissertation (MSc (Genetics))--University of Pretoria, 2009.
Genetics
unrestricted

The helicase domain of the hepatitis C virus (HCV) protein NS3 is a major immunodominant region that elicits a strong and specific immune response in the majority of HCV infections. Use of NS3 helicase-derived antigens in immunometric diagnostic assays is a mainstay in detection of HCV infections. However, thermal instability of NS3 helicase-related polypeptides is a common and severe issue hampering reproducibility, performances and shelf-life of HCV diagnostic immunoassays. In this work, a novel NS3 helicase-derived antigen is presented, with a sensible improvement in thermal stability and diagnostic performances over the currently used NS3 helicase-related antigens.

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Calves born persistently infected (PI) with non-cytopathic bovine viral diarrhea virus (ncpBVDV) frequently develop a fatal gastroenteric illness called mucosal disease (MD). From animals affected by MD, both the original virus (ncpBVDV) and an antigenically identical, yet cytopathic virus (cpBVDV) can be isolated. Cytopathic BVDVs are originated from the ncp counterparts by diverse genetic mechanisms, all leading to the expression of the non-structural polypeptide NS3 as a discrete protein. In contrast, ncpBVDVs express only the large precursor polypeptide, NS2-3, which contains the NS3 sequence within its carboxy-terminal half. The investigation of the mechanism leading to NS3 expression in 41 cpBVDV isolates is reported. An RT-PCR strategy was designed to detect insertions within the NS2-3 gene and/or duplication of the NS3 gene two common mechanisms of expression of NS3. RT-PCR amplification revealed insertions in the NS2-3 gene of three cp isolates, being the inserts similar in size to that present in the cpBVDV NADL strain. Sequencing of one such insert revealed a 296 nucleotide sequence, with a central core of 270 putative aminoacid sequence highly homologous (98%) to the NADL insert, a sequence corresponding to the cellular J-Domain gene. Another cpBVDV isolate contained a duplication of the NS3 gene downstream from the original locus. In contrast, no detectable NS2-3 insertions nor NS3 gene duplications were observed in the genome of 37 cp isolates. These results demonstrate that cleavage of NS2-3 without bulk RNA insertions nor NS3 gene duplications seems to be a frequent mechanism leading to NS3 expression and BVDV cytopathology.
Bezerros nascidos persistentemente infectados (PI) com o biótipo não citopático (ncp) do vírus da Diarréia Viral Bovina (BVDV) freqüentemente desenvolvem uma doença gastroentérica fatal, chamada de Doença das Mucosas (DM). Dos animais afetados pela DM é possível isolar o vírus original não-citopático (BVDVncp) e um vírus antigenicamente idêntico, porém citopático (BVDVcp). Os BVDVcps são gerados a partir do vírus original ncp por diversos mecanismos genéticos, que resultam na expressão da proteína não-estrutural NS3 como uma proteína individual. Em contrapartida, os BVDVncp expressam somente a proteína precursora NS2-3, que contém a seqüência da NS3 no seu terço carboxi-terminal. Este trabalho relata a investigação dos mecanismos genéticos associados com a expressão da NS3 em 41 isolados citopáticos de BVDV. Uma estratégia de RT-PCR foi delineada para detectar inserções no gene da NS2-3 e/ou duplicações no gene da NS3, dois mecanismos freqüentes de expressão da NS3. Amplificação do genoma dos 41 isolados por RT-PCR revelou a presença de inserções no gene da NS2-3 em três isolados, de tamanho similar a inserção presente na cepa de BVDVcp NADL. O seqüenciamento da inserção de um isolado revelou uma seqüência de 296 nucleotídeos, com uma região central de 270 nucleotídeos altamente homóloga (98%) com a inserção da cepa NADL, que corresponde a uma seqüência do gene celular J-Domain. Outro isolado de BVDVcp contém uma duplicação do gene da NS3 na direção 3 da sua posição original. Em 37 isolados cp não foram detectadas inserções na NS2-3 ou duplicações da NS3. Esses resultados demonstram que a clivagem da NS2-3 sem a presença de inserções de RNA ou duplicações do gene da NS3 parecem ser mecanismos frequentes de expressão da proteína NS3 e citopatologia no BVDV.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated.

Report code: LIU-TEK-LIC-2006:46.

The aims of this investigation were to compare the nonstructural proteins, NS3 and NS3A, of African horse sickness virus (AHSV) and to further characterize the cytotoxic properties of these proteins. NS3 and NS3A are encoded from two in-phase overlapping reading frames on the smallest double-stranded (ds) RNS genome segment, segment 10 (S10) of AHSV. The proteins differ only with respect to the presence of an additional 10 or 11 amino acids, depending on the serotype, at the N-terminal of NS3. All known orbiviruses have been shown to encode two closely related proteins from S10. Sequence analysis of the N-terminal region of the NS3 proteins of the different serotypes of AHSV and various orbiviruses revealed that this region is not highly conserver. Both AHSV NS3 and NS3A are membrane-associated and cytotoxic to insect cells causing membrane permeabilisation and eventual cell death when expressed individually (Van Staden et al., 1995; Van Staden et al., 1998; Van Niekerk et al., 2001a). AHSV infection of Vero cells results in the synthesis of both proteins in equimolar amounts (Van Staden, 1993). The effect on the cytotoxic properties of these proteins when expressed together in insect cells was therefore investigated here. Whether co-expressed or expressed individually NS3 and NS3A caused a dramatic decrease in the viability of insect cells. The NS3 protein is therefore representative of the NS3 and NS3A proteins together in terms of its cytotoxic effect. The effect of the exogenous addition of NS3 on the membrane permeability of Vero cells was also investigated. The NS3 protein was found to cause a rapid increase in the membrane permeability of Vero cells. The cytotoxic properties of NS3 appear therefore not to be limited to their endogenous effects on insect cells. The AHSV-3 NS3 and NS3A proteins were expressed as histidine tagged recombinants in the baculovirus expression system, to allow for the purification of large quantities of protein for functional and comparative studies. The resulting NS3 histidine fusion product, however, displayed a decrease in solubility, probably as a result of incorrect folding due to the presence of this histidine tag extension at the N-terminus of the protein. To produce antibodies that detect NS32, and not NS3A, in AHSV infected cells, the N-terminal region unique to AHSV-3 NS3 was displayed on the surface of the AHSV core protein, VPO7. The chimeric protein VP7-NS3 displayed the same structural characteristics as the wild type VP7 protein, aggregating into highly insoluble crystals. Antiserum was prepared against purified VP7-NS3 and analysed in terms of its ability to recognize denatured and non-denatured AHSV-3 NS3. Although the antiserum was shown to contain antibodies directed against VP7 epitopes no immune reaction with NS3 was observed. The use of alternate sites on the surface region of VP7 for the display of such a small peptide needs to be investigated. Although no functional differences between NS3 and NS3A were identified in this investigation, the finding that NS3 causes membrane permeability of damage to Vero cells represents the first indication that this AHSV protein causes extra cellular membrane damage in mammalian cells. Many viral membrane damaging proteins or viroporins are thought to contribute significantly to the severity of virus-induced pathogenesis. The mechanism of membrane damage and the contribution of the membrane damaging properties of NS3 to AHSV-induced pathogenesis needs to be investigated
Dissertation (MSc (Genetics))--University of Pretoria, 2006.
Genetics
unrestricted

碩士
臺北醫學大學
細胞及分子生物研究所
93
The C terminal domain of dengue virus nonstructural protein 3 (NS3) contains the conserved motifs present in several RNA helicases and has NTPase, RNA triphosphotase, and RNA helicase activities. In this research, we expressed and purified a His-tag fusion protein containing the C terminal domain of dengue virus NS3 (named as NS3H). In addition, we constructed and prepared the substitution mutants of NS3H. We studied the ATPase activity of the wild type and the mutant proteins, and examined the relationship among NS3H, ATP, and RNA. The ATPase activity required divalent cation to function. Mg2+ was more effective than Mn2+ and Ca2+ in catalyzing ATP hydrolysis, while Mn2+ strongly inhibited the stimulatory effect of Mg2+. Thus, MnATP appears to have a higher affinity to NS3H than that of MgATP. Both NaCl and rU35 (U homopolymer of 35 nt long) accelerated the rate of ATP hydrolysis, but they did not alter the Km to ATP (90-120 µM). The increase of NaCl concentration from 10 mM to 30 mM eliminated the stimulatory effect of rU35 on ATP hydrolysis, suggesting the interaction between rU35 and NS3H was mainly electrostatic. The K199A, T200A substitutions in the motif I as well as the D284A substitution in the motif II abolished the ATPase activity of NS3H, while the D284E substitution mutant possessed the RNA stimulated ATPase activity. These results reveal that the motifs I and II participate in ATP binding and ATP hydrolysis. M429 and W488 were the “gatekeepers” of the conserved RNA helicase domain. The M429A substitution mutant interacted with ATP and rU35 in a manner similar to the wild type protein, while it hydrolyzed ATP less efficiently than that of NS3H. The W488A substitution mutant had a very low ATPase activity. Instead of being stimulated by rU35, the ATPase activity of this mutant was inhibited by rU35, and the inhibitory effect of rU35 was attenuated when the NaCl concentration increased. Thus, the substitution at each gatekeeper may not affect the RNA binding activity of NS3H. In summary, the results of this study suggest that the binding of RNA to NS3H may induce the conformation rearrangement of NS3H that increases the ATP hydrolysis rate but does not alter the Km to ATP. The gatekeeper mutants may have defect in the conformation rearrangement process, as a result the ATPase activity is attenuated.

碩士
國立東華大學
資訊工程學系
107
The network slicing and packet scheduling are the key mechanisms to provide Quality of Service and user requirements in computer network. The network slicing can support minimum bandwidth guarantee and let operators provide portions of their networks for specific customer uses cases. The packet scheduling can allocate the resource according to the user requirements, and support the thourthput fairness between the service. In the paper, we concern about the thourthput fairness between the different kind of packet scheduling algorithms. The different kind of packet scheduling algorithms and multi class service were implemented in Network Simulator 3. The simluation resoults show the thourthput fairness between the different kind of packet scheduling algorithms and the different kind of service.