Investigación sobre las causas que pudieron originar el hundimiento de la cubierta del tercer depósito del Canal de Isabel II en 1905

En la presente investigación se analiza la causa del hundimiento del cuarto compartimento del Tercer Depósito del Canal de Isabel II el 8 de abril de 1905, uno de los más graves de la historia de la construcción en España: fallecieron 30 personas y quedaron heridas otras 60. El Proyecto y Construcción de esta estructura era de D. José Eugenio Ribera, una de las grandes figuras de la ingeniería civil en nuestro país, cuya carrera pudo haber quedado truncada como consecuencia del siniestro. Dado el tiempo transcurrido desde la ocurrencia de este accidente, la investigación ha partido de la recopilación de la información relativa al Proyecto y a la propia construcción de la estructura, para revisar a continuación la información disponible sobre el hundimiento. De la construcción de la cubierta es interesante destacar la atrevida configuración estructural, cubriéndose una inmensa superficie de 74.000 m2 mediante una sucesión de bóvedas de hormigón armado de tan sólo 5 cm de espesor y un rebajamiento de 1/10 para salvar una luz de 6 m, que apoyaban en pórticos del mismo material, con pilares también muy esbeltos: 0,25 m de lado para 8 m de altura. Y todo ello en una época en la que la tecnología y conocimiento de las estructuras con este "nuevo" material se basaban en buena medida en el desarrollo de patentes. En cuanto a la información sobre el hundimiento, llama la atención en primer lugar la relevancia de los técnicos, peritos y letrados que intervinieron en el juicio y en el procedimiento administrativo posterior, poniéndose de manifiesto la trascendencia que el accidente tuvo en su momento y que, sin embargo, no ha trascendido hasta nuestros días. Ejemplo de ello es el papel de Echegaray -primera figura intelectual de la época- como perito en la defensa de Ribera, de D. Melquiades Álvarez -futuro presidente del Congreso- como abogado defensor, el General Marvá -uno de los máximos exponentes del papel de los ingenieros militares en la introducción del hormigón armado en nuestro país-, que presidiría la Comisión encargada del peritaje por parte del juzgado, o las opiniones de reconocidas personalidades internacionales del "nuevo" material como el Dr. von Emperger o Hennebique. Pero lo más relevante de dicha información es la falta de uniformidad sobre lo que pudo ocasionar el hundimiento: fallos en los materiales, durante la construcción, defectos en el diseño de la estructura, la realización de unas pruebas de carga cuando se concluyó ésta, etc. Pero la que durante el juicio y en los Informes posteriores se impuso como causa del fallo de la estructura fue su dilatación como consecuencia de las altas temperaturas que se produjeron aquella primavera. Y ello a pesar de que el hundimiento ocurrió a las 7 de la mañana... Con base en esta información se ha analizado el comportamiento estructural de la cubierta, permitiendo evaluar el papel que diversos factores pudieron tener en el inicio del hundimiento y en su extensión a toda la superficie construida, concluyéndose así cuáles fueron las causas del siniestro. De los resultados obtenidos se presta especial atención a las enseñanzas que se desprenden de la ocurrencia del hundimiento, enfatizándose en la relevancia de la historia -y en particular de los casos históricos de error- para la formación continua que debe existir en la Ingeniería. En el caso del hundimiento del Tercer Depósito algunas de estas "enseñanzas" son de plena actualidad, tales como la importancia de los detalles constructivos en la "robustez" de la estructuras, el diseño de estructuras "integrales" o la vigilancia del proceso constructivo. Por último, la investigación ha servido para recuperar, una vez más, la figura de D. José Eugenio Ribera, cuyo papel en la introducción del hormigón armado en España fue decisivo. En la obra del Tercer Depósito se arriesgó demasiado, y provocó un desastre que aceleró la transición hacia una nueva etapa en el hormigón estructural al abrigo de un mayor conocimiento científico y de las primeras normativas. También en esta etapa sería protagonista. This dissertation analyses the cause of the collapse of the 4th compartment of the 3th Reservoir of Canal de Isabel II in Madrid. It happened in 1905, on April 8th, being one of the most disastrous accidents occurred in the history of Spanish construction: 30 people died and 60 were injured. The design and construction supervision were carried out by D. José Eugenio Ribera, one of the main figures in Civil Engineering of our country, whose career could have been destroyed as a result of this accident. Since it occurred more than 100 years ago, the investigation started by compiling information about the structure`s design and construction, followed by reviewing the available information about the accident. With regard to the construction, it is interesting to point out its daring structural configuration. It covered a huge area of 74.000 m2 with a series of reinforced concrete vaults with a thickness of not more than 5 cm, a 6 m span and a rise of 1/10th. In turn, these vaults were supported by frames composed of very slender 0,25 m x 0,25 m columns with a height of 8 m. It is noteworthy that this took place in a time when the technology and knowledge about this "new" material was largely based on patents. In relation to the information about the collapse, its significance is shown by the important experts and lawyers that were involved in the trial and the subsequent administrative procedure. For example, Echegaray -the most important intellectual of that time- defended Ribera, Melquiades Álvarez –the future president of the Congress- was his lawyer, and General Marvá -who represented the important role of the military engineers in the introduction of reinforced concrete in our country-, led the Commission that was put in charge by the judge of the root cause analysis. In addition, the matter caught the interest of renowned foreigners like Dr. von Emperger or Hennebique and their opinions had a great influence. Nonetheless, this structural failure is unknown to most of today’s engineers. However, what is most surprising are the different causes that were claimed to lie at the root of the disaster: material defects, construction flaws, errors in the design, load tests performed after the structure was finished, etc. The final cause that was put forth during the trial and in the following reports was attributed to the dilatation of the roof due to the high temperatures that spring, albeit the collapse occurred at 7 AM... Based on this information the structural behaviour of the roof has been analysed, which allowed identifying the causes that could have provoked the initial failure and those that could have led to the global collapse. Lessons have been learned from these results, which points out the relevance of history -and in particular, of examples gone wrong- for the continuous education that should exist in engineering. In the case of the 3th Reservoir some of these lessons are still relevant during the present time, like the importance of detailing in "robustness", the design of "integral" structures or the due consideration of construction methods. Finally, the investigation has revived, once again, the figure of D. José Eugenio Ribera, whose role in the introduction of reinforced concrete in Spain was crucial. With the construction of the 3th Reservoir he took too much risk and caused a disaster that accelerated the transition to a new era in structural concrete based on greater scientific knowledge and the first codes. In this new period he would also play a major role.

Unwinding of nucleic acids by HCV NS3 helicase is sensitive to the structure of the duplex

Hepatitis C virus (HCV) helicase, non-structural protein 3 (NS3), is proposed to aid in HCV genome replication and is considered a target for inhibition of HCV. In order to investigate the substrate requirements for nucleic acid unwinding by NS3, substrates were prepared by annealing a 30mer oligonucleotide to a 15mer. The resulting 15 bp duplex contained a single-stranded DNA overhang of 15 nt referred to as the bound strand. Other substrates were prepared in which the 15mer DNA was replaced by a strand of peptide nucleic acid (PNA). The PNA–DNA substrate was unwound by NS3, but the observed rate of strand separation was at least 25-fold slower than for the equivalent DNA–DNA substrate. Binding of NS3 to the PNA–DNA substrate was similar to the DNA–DNA substrate, due to the fact that NS3 initially binds to the single-stranded overhang, which was identical in each substrate. A PNA–RNA substrate was not unwound by NS3 under similar conditions. In contrast, morpholino–DNA and phosphorothioate–DNA substrates were utilized as efficiently by NS3 as DNA–DNA substrates. These results indicate that the PNA–DNA and PNA–RNA heteroduplexes adopt structures that are unfavorable for unwinding by NS3, suggesting that the unwinding activity of NS3 is sensitive to the structure of the duplex.

Automatização do dimensionamento de elementos estruturais em betão armado com GFRP

A presente dissertação teve como objetivo o desenvolvimento de uma aplicação informática para automatização do dimensionamento de elementos de betão armado com GFRP, através da utilização de guias e normas existentes. O documento elaborado apresenta uma breve síntese da situação atual do estado do conhecimento (estado da arte) nesta temática, uma abordagem aos conceitos de dimensionamento, de elementos estruturais baseados em documentos normativos, e alguns conceitos sobre a programação efetuada, quer através da descrição das metodologias quer por esquematização em figuras. Neste documento é, também, descrito o funcionamento geral do programa, exemplos de aplicação prática e breves considerações sobre o trabalho desenvolvido. A aplicação efetua o cálculo de vigas e lajes de acordo com o guia do Committee 440 do American Concrete Institute, ACI 440.1R-06 – “Guide for the Design and Construction of Structural Concrete Reinforced with FRP Bars”, bem como através de uma análise não linear, como descrito na norma NP EN 1992: Eurocódigo 2 – “Projecto de estruturas de betão”. Foram ainda realizados cálculos para determinação de diagramas de interação de flexão composta, com pares de esforços axiais e de flexão, utilizados para o dimensionamento de colunas. No final do desenvolvimento da aplicação o programa foi testado com exemplos de dimensionamento, para que fosse possível realizar uma análise crítica aos resultados obtidos e ao próprio modo de funcionamento da aplicação.

Wrapping things up about virus RNA replication

All single-stranded 'positive-sense' RNA viruses that infect mammalian, insect or plant cells rearrange internal cellular membranes to provide an environment facilitating virus replication. A striking feature of these unique membrane structures is the induction of 70-100 nm vesicles (either free within the cytoplasm, associated with other induced vesicles or bound within a surrounding membrane) harbouring the viral replication complex (RC). Although similar in appearance, the cellular composition of these vesicles appears to vary for different viruses, implying different organelle origins for the intracellular sites of viral RNA replication. Genetic analysis has revealed that induction of these membrane structures can be attributed to a particular viral gene product, usually a non-structural protein. This review will highlight our current knowledge of the formation and composition of virus RCs and describe some of the similarities and differences in RNA-membrane interactions observed between the virus families Flaviviridae and Picornaviridae.

Relationship of body condition score and blood urea and ammonia to pregnancy in Italian Mediterranean buffaloes

The relationship of body condition score ( BCS) and blood urea and ammonia to pregnancy outcome was examined in Italian Mediterranean Buffalo cows mated by AI. The study was conducted on 150 buffaloes at 145 +/- 83 days in milk that were fed a diet comprising 14.8% crude protein, 0.9 milk forage units . kg(-1) dry matter and a non- structural carbohydrate/ crude protein ratio of 2.14. The stage of the oestrous cycle was synchronised by the Ovsynch- TAI programme and blood urea and ammonia levels were assessed on the day of AI. Energy corrected milk ( ECM) production and BCS were recorded bi- weekly. The pregnancy risk was 46.7% and was slightly lower in buffaloes with BCS < 6.0 and BCS > 7.5. There were no significant differences in ECM, urea and ammonia between pregnant and non- pregnant buffaloes. However, pregnancy outcome was higher ( P = 0.02) in buffaloes with blood urea < 6.83 mmol . L-1. The likelihood of pregnancy for buffaloes with low urea blood level was 2.6 greater than for high urea level and exposure to a high urea level lowered the probability of pregnancy by about 0.25. The findings indicate that buffaloes are similar to cattle and increased blood levels of urea are associated with reduced fertility when animals are mated by AI.

An energy based nanomechnical properties evaluation method for cementitious materials

Advances in multiscale material modeling of structural concrete have created an upsurge of interest in the accurate evaluation of mechanical properties and volume fractions of its nano constituents. The task is accomplished by analyzing the response of a material to indentation, obtained as an outcome of a nanoindentation experiment, using a procedure called the Oliver and Pharr (OP) method. Despite its widespread use, the accuracy of this method is often questioned when it is applied to the data from heterogeneous materials or from the materials that show pile-up and sink-in during indentation, which necessitates the development of an alternative method. ^ In this study, a model is developed within the framework defined by contact mechanics to compute the nanomechanical properties of a material from its indentation response. Unlike the OP method, indentation energies are employed in the form of dimensionless constants to evaluate model parameters. Analysis of the load-displacement data pertaining to a wide range of materials revealed that the energy constants may be used to determine the indenter tip bluntness, hardness and initial unloading stiffness of the material. The proposed model has two main advantages: (1) it does not require the computation of the contact area, a source of error in the existing method; and (2) it incorporates the effect of peak indentation load, dwelling period and indenter tip bluntness on the measured mechanical properties explicitly. ^ Indentation tests are also carried out on samples from cement paste to validate the energy based model developed herein by determining the elastic modulus and hardness of different phases of the paste. As a consequence, it has been found that the model computes the mechanical properties in close agreement with that obtained by the OP method; a discrepancy, though insignificant, is observed more in the case of C-S-H than in the anhydrous phase. Nevertheless, the proposed method is computationally efficient, and thus it is highly suitable when the grid indentation technique is required to be performed. In addition, several empirical relations are developed that are found to be crucial in understanding the nanomechanical behavior of cementitious materials.^

Estudo in silico das interações da protease NS3-NS2B de DENV-2 com o inibidor peptídico Bz-nKRR-H com finalidades terapêuticas

Dengue virus is an important patogen that causes Dengue desease in all world, and belongs to Flavivirus gender. The virus consists of enveloped RNA with a single strand positive sense, 11Kb genome. The RNA is translated into a polyprotein precursor, wich is cleaved into 3 structural proteins (C, prM e E) and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5). The NS3 is a multifunctional protein, that besides to promote the polyprotein precursor cleavage, also have NTPase, helicase and RTPase activity. The NS3 needs a hydrophilic segment of 40 residues from the transmembrane NS2B protein (who acts like cofator) to realize this functions. Actually, there's no vacines available on the market, and the treatment are just symptomatic. The tetrapeptide inhibitor Bz-Nle-Lys-Arg-Arg-H (Ki de 5,8-7,0 M) was showed as a potent inhibitor μ for NS3prot in Dengue virus. That is a inteligent alternative to treat the dengue desease. The present work aimed analyse the interactions of the ligand bounded to the activity site to provid a clear and depth vision of that interaction. For this purpouse, it was conducted an in silico study, by using quantum mechanical calculations based on Density Functional Theory (DFT), with Generalized Gradient approximation (GGA) to describe the effects of exchange and correlation. The interaction energy of each amino acid belonging to the binding site to the ligand was calculated the using the method of molecular fragmentation with conjugated caps (MFCC). Besides energy, we calculated the distances, types of molecular interactions and atomic groups involved. The theoretical models used were satisfactory and show a more accurate description when the dielectric constant = 20 ε and 80 was used. The results demonstrate that the interaction energy of the system reached convergence at 13.5 A. Within a radius of 13,5A the most important residues were identified. Met49, Met84 and Asp81 perform interactions of hydrogen with the ligant. The Asp79 and Asp75 residues present high energy of attraction. Arg54, Arg85 and Lys 131 perform hydrogen interactions with the ligand, however, appear in BIRD graph having high repulsion energy with the inhibitor. The data also emphasizes the importance of residue Tyr161 and the involvement of the catalytic triad composed by Asp75, His51 and Ser135

Crescimento urbano e seus impactos no sistema de drenagem de uma bacia em Natal/RN

The uncontrolled growth of most Brazilian cities is not accompanied by the development of urban infrastructure. With increasing soil sealing, runoff and decreased infiltration volume, impacts on water resources and on population of the areas affected by urban growth are inevitable. This study aims to evaluate the use and occupation of a watershed and analyze the drainage system in order to control the impact using tools to integrate urban development with the drainage of rainwater in an important watershed in the Natal City, Rio Grande do Norte State. The study involved the characterization of the basin XII.4 on the land use and occupation, for the years 2005 and 2014. With the application of SWMM model was possible to analyze the impacts caused by the urbanization process in the existing drainage system, showing the two years analyzed have their areas very close to percentage saturation of impervious areas. Although the region is still predominantly single family residential, suffers increasing verticalization of mainly commercial buildings. The drainage system is inefficient for the area's needs by the year 2005. The drainage system was also tested for four variations of land use by developing scenarios. Scenario 1 is related to the year 2014, considered current. Scenario 2 was adopted the maximum rate of 80% for land use, allowed for the Natal City. Scenario 3 gives the critical condition of land use, with the area 100% impervious. Scenario 4 is applied to the existence of LID (Low Impact Device). The scenarios analysis showed that all indicate deficiency at some point of the drainage system as a result of the high degree of occupation of the area that generate higher flows than the initial drainage system capacity. With the study it became clear that the adoption of non-structural tools are effective in reducing flooding and improving the drainage system capacity.

Crescimento urbano e seus impactos no sistema de drenagem de uma bacia em Natal/RN

The uncontrolled growth of most Brazilian cities is not accompanied by the development of urban infrastructure. With increasing soil sealing, runoff and decreased infiltration volume, impacts on water resources and on population of the areas affected by urban growth are inevitable. This study aims to evaluate the use and occupation of a watershed and analyze the drainage system in order to control the impact using tools to integrate urban development with the drainage of rainwater in an important watershed in the Natal City, Rio Grande do Norte State. The study involved the characterization of the basin XII.4 on the land use and occupation, for the years 2005 and 2014. With the application of SWMM model was possible to analyze the impacts caused by the urbanization process in the existing drainage system, showing the two years analyzed have their areas very close to percentage saturation of impervious areas. Although the region is still predominantly single family residential, suffers increasing verticalization of mainly commercial buildings. The drainage system is inefficient for the area's needs by the year 2005. The drainage system was also tested for four variations of land use by developing scenarios. Scenario 1 is related to the year 2014, considered current. Scenario 2 was adopted the maximum rate of 80% for land use, allowed for the Natal City. Scenario 3 gives the critical condition of land use, with the area 100% impervious. Scenario 4 is applied to the existence of LID (Low Impact Device). The scenarios analysis showed that all indicate deficiency at some point of the drainage system as a result of the high degree of occupation of the area that generate higher flows than the initial drainage system capacity. With the study it became clear that the adoption of non-structural tools are effective in reducing flooding and improving the drainage system capacity.

Updating of an instrumented building model considering amplitude dependence of dynamic resonant properties extracted from seismic response records

The authors would like to express their gratitude to their supporters. Drs Jim Cousins, S.R. Uma and Ken Gledhill facilitated this research by providing access to GeoNet seismic data and structural building information. Piotr Omenzetter’s work within the Lloyd’s Register Foundation Centre for Safety and Reliability Engineering at the University of Aberdeen is supported by Lloyd’s Register Foundation. The Foundation helps to protect life and property by supporting engineering-related education, public engagement and the application of research.

Updating of an instrumented building model considering amplitude dependence of dynamic resonant properties extracted from seismic response records

The authors would like to express their gratitude to their supporters. Drs Jim Cousins, S.R. Uma and Ken Gledhill facilitated this research by providing access to GeoNet seismic data and structural building information. Piotr Omenzetter’s work within the Lloyd’s Register Foundation Centre for Safety and Reliability Engineering at the University of Aberdeen is supported by Lloyd’s Register Foundation. The Foundation helps to protect life and property by supporting engineering-related education, public engagement and the application of research.

First complete genome sequence of a capsicum chlorosis tospovirus isolate from Australia with an unusually large S RNA intergenic region

The first complete genome sequence of capsicum chlorosis virus (CaCV) from Australia was determined using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV had a tripartite genome structure like other CaCV isolates. The large (L) RNA was 8913 nucleotides (nt) in length and contained a single open reading frame (ORF) of 8634 nt encoding a predicted RNA-dependent RNA polymerase (RdRp) in the viral-complementary (vc) sense. The medium (M) and small (S) RNA segments were 4846 and 3944 nt in length, respectively, each containing two non-overlapping ORFs in ambisense orientation, separated by intergenic regions (IGR). The M segment contained ORFs encoding the predicted non-structural movement protein (NSm; 927 nt) and precursor of glycoproteins (GP; 3366 nt) in the viral sense (v) and vc strand, respectively, separated by a 449-nt IGR. The S segment coded for the predicted nucleocapsid (N) protein (828 nt) and non-structural suppressor of silencing protein (NSs; 1320 nt) in the vc and v strand, respectively. The S RNA contained an IGR of 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. Comparison of the Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with a Taiwanese isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for at least two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses, separate from CaCV.

Molecular and biological characterisation of orthobunyaviruses

Orthobunyaviruses are the largest genus within the Bunyaviridae family, with over 170 named viruses classified into 18 serogroups (Elliott and Blakqori, 2001; Plyusnin et al., 2012). Orthobunyaviruses are transmitted by arthropods and have a tripartite negative sense RNA genome, which encodes 4 structural proteins and 2 non-structural proteins. The non-structural protein NSs is the primary virulence factor of orthobunyaviruses and potent antagonist of the type I interferon (IFN) response. However, sequencing studies have identified pathogenic viruses that lack the NSs protein (Mohamed et al., 2009; Gauci et al., 2010). The work presented in this thesis describes the molecular and biological characterisation of divergent orthobunyaviruses. Data on plaque morphology, growth kinetics, protein profiles, sensitivity to IFN and activation of the type I IFN system are presented for viruses in the Anopheles A, Anopheles B, Capim, Gamboa, Guama, Minatitlan, Nyando, Tete and Turlock serogroups. These are complemented with complete genome sequencing and phylogenetic analysis. Low activation of IFN by Tete serogroup viruses, which naturally lack an NSs protein, was also further investigated by the development of a reverse genetics system for Batama virus (BMAV). Recombinant viruses with mutations in the virus nucleocapsid protein amino terminus showed higher activation of type I IFN in vitro and data suggests that low levels of IFN are due to lower activation rather than active antagonism. The anti-orthobunyavirus activity of IFN-stimulated genes IFI44, IFITMs and human and ovine BST2 were also studied, revealing that activity varies not only within the orthobunyavirus genus and virus serogroups but also within virus species. Furthermore, there was evidence of active antagonism of the type I IFN response and ISGs by non-NSs viruses. In summary, the results show that pathogenicity in man and antagonism of the type I IFN response in vitro cannot be predicted by the presence, or absence, of an NSs ORF. They also highlight problems in orthobunyavirus classification with discordance between classical antigen based data and phylogenetic analysis.

Efficiency of rumen microbial protein synthesis in cattle grazing tropical pastures as estimated by a novel technique

The efficiency of microbial protein synthesis (EMPS) in cattle grazing a range of tropical pasture types was examined using a new method of intra-jugular infusion of CrEDTA to estimate urinary excretion of purine derivatives (PD). Seven pasture types were studied in south-east Queensland, Australia, over a 13-month period. These included native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii) studied in the early wet, the wet/dry transition and the dry season; introduced tropical grass (C4) pasture (Bothriochloa insculpta) in the mid wet season; two introduced tropical legume species (C3), (Lablab purpureus and Clitoria ternatea); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum). There was a large range in EMPS across pasture types: 26-209 g microbial crude protein (MCP)/kg digestible organic matter intake (DOMI). Estimated rumen degradable protein (RDP) supply (42-525 g/kg DOMI) was the major factor associated with EMPS across the range of pasture types studied. EMPS in steers grazing all tropical grass pastures was low (<130 g/kg DOMI) and limited by RDP supply. Negative linear relationships (P<0.05) between EMPS and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in extrusa were evident. However, non-fibre carbohydrate in extrusa, total non-structural carbohydrate concentration in plucked pasture leaf, rumen fluid and particle dilution rate, protozoal concentration in rumen fluid and rumen fluid pH were not correlated with EMPS. It was concluded that EMPS was well below 130 g MCP/kg DOMI when cattle grazed unfertilised, tropical grass pastures in south-east Queensland and that RDP was the primary limiting nutrient. High EMPS was associated with very high RDP, vastly in excess of RDP requirements by microbes

Host cellular regulatory networks in dengue virus-human interactions

Dengue fever is one of the most important mosquito-borne diseases worldwide and is caused by infection with dengue virus (DENV). The disease is endemic in tropical and sub-tropical regions and has increased remarkably in the last few decades. At present, there is no antiviral or approved vaccine against the virus. Treatment of dengue patients is usually supportive, through oral or intravenous rehydration, or by blood transfusion for more severe dengue cases. Infection of DENV in humans and mosquitoes involves a complex interplay between the virus and host factors. This results in regulation of numerous intracellular processes, such as signal transduction and gene transcription which leads to progression of disease. To understand the mechanisms underlying the disease, the study of virus and host factors is therefore essential and could lead to the identification of human proteins modulating an essential step in the virus life cycle. Knowledge of these human proteins could lead to the discovery of potential new drug targets and disease control strategies in the future. Recent advances of high throughput screening technologies have provided researchers with molecular tools to carry out investigations on a large scale. Several studies have focused on determination of the host factors during DENV infection in human and mosquito cells. For instance, a genome-wide RNA interference (RNAi) screen has identified host factors that potentially play an important role in both DENV and West Nile virus replication (Krishnan et al. 2008). In the present study, a high-throughput yeast two-hybrid screen has been utilised in order to identify human factors interacting with DENV non-structural proteins. From the screen, 94 potential human interactors were identified. These include proteins involved in immune signalling regulation, potassium voltage-gated channels, transcriptional regulators, protein transporters and endoplasmic reticulum-associated proteins. Validation of fifteen of these human interactions revealed twelve of them strongly interacted with DENV proteins. Two proteins of particular interest were selected for further investigations of functional biological systems at the molecular level. These proteins, including a nuclear-associated protein BANP and a voltage-gated potassium channel Kv1.3, both have been identified through interaction with the DENV NS2A. BANP is known to be involved in NF-kB immune signalling pathway, whereas, Kv1.3 is known to play an important role in regulating passive flow of potassium ions upon changes in the cell transmembrane potential. This study also initiated a construction of an Aedes aegypti cDNA library for use with DENV proteins in Y2H screen. However, several issues were encountered during the study which made the library unsuitable for protein interaction analysis. In parallel, innate immune signalling was also optimised for downstream analysis. Overall, the work presented in this thesis, in particular the Y2H screen provides a number of human factors potentially targeted by DENV during infection. Nonetheless, more work is required to be done in order to validate these proteins and determine their functional properties, as well as testing them with infectious DENV to establish a biological significance. In the long term, data from this study will be useful for investigating potential human factors for development of antiviral strategies against dengue.