Os processos de cheias em bacias urbanas vêm sendo agravados devido a uma série de motivos associados à ocupação desordenada do solo urbano. As medidas de controle de enchentes podem ter sua eficiência analisada através da modelagem hidrológica matemática. É neste sentido que o presente trabalho analisa a influência da urbanização sobre os distúrbios no escoamento superficial, por meio de simulações de cenários urbanísticos propostos, com a finalidade de servir como ferramenta de planejamento urbano. Para isso, definiu-se como área de estudo, parte da bacia do Córrego do Gregório, São Carlos - SP. As principais informações da bacia a serem consideradas são: topografia, hidrografia, uso do solo urbano, expansão da área urbana, áreas de proteção ambiental e divisores de microbacias. O modelo hidrológico utilizado é o IPHS-1, do tipo concentrado. Para essa análise são propostos e simulados cenários urbanísticos, baseados na adoção de medidas de controle de inundações não-estruturais, referindo-se principalmente, à conservação de áreas verdes e disciplinamento do uso e ocupação do solo, verificando sua eficiência na redução do volume escoado e atenuação das vazões de pico. / The flooding processes in urban basins have become worse due to many reasons. All of them associated with the disorganized occupancy of the urban area land. The efficiency of flood mitigation measures can be analyzed by mathematical modeling. This study aims to be used as a tool for urban planning and it analyses the influence of the urbanization processes on surface runoff, using simulation of several urbanization scenarios. The case study was undertaken at the Gregório River Basin in São Carlos - SP. The main information considered was topography, hydrology, urban land use, urbanization, protected land and sub-basins. The software used was IPHS-1 which is a lumped hydrologic model. In this research many urbanization scenarios are proposed and simulated. These views are based in many nonstructural flood mitigation alternatives such as land cover conservation, use and occupancy of the land, in order to check their efficiency in reducing the total volume of surface runoff and the peak flow.

Os processos de cheias em bacias urbanas vêm sendo agravados devido a uma série de motivos associados à ocupação desordenada do solo urbano. As medidas de controle de enchentes podem ter sua eficiência analisada através da modelagem hidrológica matemática. É neste sentido que o presente trabalho analisa a influência da urbanização sobre os distúrbios no escoamento superficial, por meio de simulações de cenários urbanísticos propostos, com a finalidade de servir como ferramenta de planejamento urbano. Para isso, definiu-se como área de estudo, parte da bacia do Córrego do Gregório, São Carlos - SP. As principais informações da bacia a serem consideradas são: topografia, hidrografia, uso do solo urbano, expansão da área urbana, áreas de proteção ambiental e divisores de microbacias. O modelo hidrológico utilizado é o IPHS-1, do tipo concentrado. Para essa análise são propostos e simulados cenários urbanísticos, baseados na adoção de medidas de controle de inundações não-estruturais, referindo-se principalmente, à conservação de áreas verdes e disciplinamento do uso e ocupação do solo, verificando sua eficiência na redução do volume escoado e atenuação das vazões de pico. / The flooding processes in urban basins have become worse due to many reasons. All of them associated with the disorganized occupancy of the urban area land. The efficiency of flood mitigation measures can be analyzed by mathematical modeling. This study aims to be used as a tool for urban planning and it analyses the influence of the urbanization processes on surface runoff, using simulation of several urbanization scenarios. The case study was undertaken at the Gregório River Basin in São Carlos - SP. The main information considered was topography, hydrology, urban land use, urbanization, protected land and sub-basins. The software used was IPHS-1 which is a lumped hydrologic model. In this research many urbanization scenarios are proposed and simulated. These views are based in many nonstructural flood mitigation alternatives such as land cover conservation, use and occupancy of the land, in order to check their efficiency in reducing the total volume of surface runoff and the peak flow.

Rubella virus (RUBV) is a positive-strand RNA virus and the causative agent of rubella and congenital rubella syndrome in humans. To replicate its RNA, RUBV forms membrane-associated spherules, called replication complexes (RCs), the induction of which requires the two virus nonstructural proteins (NSPs), P150 and P90. Interestingly, late in infection the NSPs form a unique cytoplasmic fiber network, similar in appearance to microtubules, the function of which is unknown. Little is known about the roles of the RUBV NSPs in forming these structures and, to this end, we scrutinized the behavior and biochemical properties of the NSPs, both after expression from plasmids and during RUBV infection, using mutagenic, biochemical and pharmacological approaches. The following findings were made: First, the precursor from which P150 and P90 are produced via an embedded protease at the C-terminus of P150, called P200, was required for initial targeting to cytoplasmic foci. P150 was the determinant of fiber formation and while P90 had no specific targeting sequences on its own, P90 sequences within P200 were required for correct targeting of P200. An alpha-helix at the N-terminus of P150 was also important for correct targeting of P200, putatively by mediating the interaction between P150 and P90 within the precursor. Second, the membrane binding domain within the NSPs was within the N-terminal ~450 amino acids of P150. P150 is in an exceptionally tight association with membranes. Third, both the N- and C-terminal regions of P150, and specifically long alpha-helices within these regions, are necessary for fiber formation. Fiber formation relied on an intact microtubule network, but neither microtubule repositioning nor dynamic movement along microtubules was required. Additionally, it was shown that microtubules were not necessary in RUBV replication. Finally, P150 fibers were not required for RUBV replication; however, it was shown that the fibers are likely important in formation of cytoplasmic extensions through which a novel system of cell-to-cell transport of viral RNA in the absence of virus particles appears to occur.

Rotaviruses, members of the family Reoviridae, are the major etiologic agents of severe, acute dehydrating diarrhea in the young of many mammalian species, including humans, calves and foals. Recent estimates indicate an annual death toll of approximately 600,000 infants due to rotavirus, besides inflicting staggering economic burden worldwide. Most of these deaths occur in the developing countries and India is estimated to account for about a quarter of these deaths. Extensive molecular epidemiology studies carried out by our laboratory have revealed many interesting aspects about rotavirus diversity in this country. Molecular epidemiology of rotaviruses causing severe diarrhea in foals in two organized farms in northern India was carried out. These foal rotaviruses exhibited 5 different electropherotypes (E), E1-E5. Strains belonging to E1, E2 and E5 exhibited G10, P6[1]; G3 and G1 type specificities. Though the E1 strains possessed genes encoding G10 and P6[1] type outer capsid proteins, unlike the G10, P8[11] type strain I321, they exhibited high reactivity with the G6-specific MAb suggesting that the uncommon combination altered the specificity of the conformation-dependent antigenic epitopes on the surface proteins. Strains belonging to electropherotypes E3 and E4 were untypeable. Sequence analysis of the VP7 gene from E4 strains (Erv92 and Erv99), revealed that they represent a new VP7 genotype, G16. Nonstructural protein 4 (NSP4) of rotavirus is a multidomainal, multifunctional protein and is the first viral enterotoxin identified. We have recently reported that the diarrhea-inducing and double-layered particle (DLP)–binding properties of NSP4 are dependent on a structurally and functionally overlapping conformational domain that is conferred by cooperation between the N- and C-terminal regions of the cytoplasmic tail (Jagannath et al., J. Virol, pp 412-425, 2006). Further, a stretch of 40 amino acids (aa) from the C-terminus is predicted to be unstructured and highly susceptible to trypsin cleavage. We examined the role of this unstructured C-terminus of Hg18 NSP4 and SA11 NSP4 on the biological properties of NSP4 using a series of deletion and substitution mutants of the conserved proline and tyrosine residues in this region. Gel filtration, CD spectroscopy and Thioflavin T binding studies showed that these mutants have altered secondary structural contents and either failed to multimerize efficiently or multimerized with altered conformation. The C-terminal ten residues appear to play a regulatory role on multimerization. Proline 168, tyrosine 166 and methionine 175 appear to be critical determinants of DLP binding activity whereas, proline 165 and tyrosine 85 and 131 appears to determine the affinity of binding to DLP in the context of NSP4 ∆N72. Deletion and substitution mutants exhibited severely reduced diarrhea inducing ability and DLP binding property. Of great biological significance is the drastic decrease in the diarrhea inducing ability of the N- and C- terminal deletion mutant ∆N94 ∆C29 that exhibited about 11,000-fold increase in DD50 than the wild type (WT) ∆N72. These studies revealed that the predicted unstructured C-terminus is an important determinant of biological properties of NSP4. Extensive efforts to crystallize the complete cytoplasmic tail (CT) of NSP4 were unsuccessful and to date, the structure of only a synthetic peptide corresponding to aa 95-135 has been reported. Our recent studies indicate that the interspecies variable regions from aa 135-141 as well as the extreme C-terminus are critical determinants of virus virulence and diarrhea-inducing ability of the protein. Here, we examined the crystallization properties of several deletion mutants and report the structure of a mutant recombinant NSP4 from symptomatic (SA11) and asymptomatic (I321) strains that lacked the N-terminal 94 and C-terminal 29 aa (NSP4: 95-146) at 1.67 Å and 2.7Å, respectively. In spite of the high-resolution data, electron density for the stretch of 9 residues from the C-terminus could not be seen suggesting its highly flexible nature. The crystal packing showed a clear empty space for this region. Extension of the unstructured C-terminus beyond aa 146 hindered crystallization under the experimental conditions. The present structure revealed significant differences from that of the synthetic peptide in the conformation of amino acids at the end of the helix as well as crystal packing owing to the additional space required to accommodate the unstructured virulence-determining region. Conformational differences in this critical region effected by the presence or absence of proline or glycine at specific positions in the unstructured C-terminus, could form the basis for the wide range of variation seen in the diarrhea-inducing ability of NSP4 from different strains in newborn mouse pups. Although symptomatic and asymptomatic strains do not generally differ in the presence or absence of the conserved prolines or glycines, they contain a few additional changes that could alter the unique conformation required for optimal biological activity. In conclusion, we demonstrate that the predicted unstructured C-terminal region is indeed highly flexible and is an important determinant of biological functions of the NSP4, mutations in which probably correlates with the virulence properties of the virus.

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 172-183). Also available on the Internet.

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.

Understanding the complex dynamics that underpin the response of structures in the occurrence of earthquakes is of paramount importance in ensuring community resilience. The operational continuity of structures is influenced by the performance of nonstructural components, also known as secondary structures. Inherent vulnerability characteristics, nonlinearities and uncertainties in their properties or in the excitation pose challenges that render their response determination as a non-straightforward task. This dissertation settles in the context of mathematical modelling and response quantification of seismically driven secondary systems. The case of bilinear hysteretic, rigid-plastic and free-standing rocking oscillators is first considered, as a representative class of secondary systems of distinct behaviour excited at a single point in the primary structure. The equations governing their full dynamic interaction with linear primary oscillators are derived with the purpose of assessing the appropriateness of simplified analysis methods where the secondary-primary feedback action is not accounted for. Analyses carried out in presence of pulse-type excitation have shown that the cascade approximation can be considered satisfactory for bilinear systems provided the secondary-primary mass ratio is adequately low and the system does not approach resonance. For the case of sliding and rocking systems, much lighter secondary systems need to be considered if the cascade analysis is to be adopted, with the validity of the approximation dictated by the selection of the input parameters. Based on the premise that decoupling is permitted, new analytical solutions are derived for the pulse driven nonlinear oscillators considered, conveniently expressing the seismic response as a function of the input parameters and the relative effects are quantified. An efficient numerical scheme for a general-type of excitation is also presented and is used in conjunction with an existing nonstationary stochastic far-field ground motion model to determine the seismic response spectra for the secondary oscillators at given site and earthquake characteristics. Prompted by the presence of uncertainty in the primary structure, and in line with the classical modal analysis, a novel approach for directly characterising uncertainty in the modal shapes, frequencies and damping ratios of the primary structure is proposed. A procedure is then presented for the identification of the model parameters and demonstrated with an application to linear steel frames with uncertain semi-rigid connections. It is shown that the proposed approach reduces the number of the uncertain input parameters and the size of the dynamic problem, and is thus particularly appealing for the stochastic assessment of existing structural systems, where partial modal information is available e.g. through operational modal analysis testing. Through a numerical example, the relative effect of stochasticity in a bi-directional seismic input is found to have a more prominent role on the nonlinear response of secondary oscillators when compared to the uncertainty in the primary structure. Further extending the analyses to the case of multi-attached linear secondary systems driven by deterministic seismic excitation, a convenient variant of the component-mode synthesis method is presented, whereby the primary-secondary dynamic interaction is accounted for through the modes of vibration of the two components. The problem of selecting the vibrational modes to be retained in analysis is then addressed for the case of secondary structures, which may possess numerous low frequency modes with negligible mass, and a modal correction method is adopted in view of the application for seismic analysis. The influence of various approaches to build the viscous damping matrix of the primary-secondary assembly is also investigated, and a novel technique based on modal damping superposition is proposed. Numerical applications are demonstrated through a piping secondary system multi-connected on a primary frame exhibiting various irregularities in plan and elevation, as well as a multi-connected flexible secondary system. Overall, this PhD thesis delivers new insights into the determination and understanding of the response of seismically driven secondary structures. The research is deemed to be of academic and professional engineering interest spanning several areas including seismic engineering, extreme events, structural health monitoring, risk mitigation and reliability analysis.

No description available.

Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.

Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied. Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease. Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy. In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.