Healing replicas in a software component replication system

Dissertação para obtenção do Grau de Mestre em Engenharia Informática

RepComp - replicated software components for improved performance

Trabalho apresentado no âmbito do Mestrado em Engenharia Informática, como requisito parcial para obtenção do grau de Mestre em Engenharia Informática

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Replication Data Management,needs and solutions: an initial evaluation of conceptual approaches for integrating heterogeneous replication study data

Replication Data Management (RDM) aims at enabling the use of data collections from several iterations of an experiment. However, there are several major challenges to RDM from integrating data models and data from empirical study infrastructures that were not designed to cooperate, e.g., data model variation of local data sources. [Objective] In this paper we analyze RDM needs and evaluate conceptual RDM approaches to support replication researchers. [Method] We adapted the ATAM evaluation process to (a) analyze RDM use cases and needs of empirical replication study research groups and (b) compare three conceptual approaches to address these RDM needs: central data repositories with a fixed data model, heterogeneous local repositories, and an empirical ecosystem. [Results] While the central and local approaches have major issues that are hard to resolve in practice, the empirical ecosystem allows bridging current gaps in RDM from heterogeneous data sources. [Conclusions] The empirical ecosystem approach should be explored in diverse empirical environments.

Will diverse Tat interactions lead to novel antiretroviral drug targets?

More than fifteen years following the description of Tat as a critical HIV gene expression regulatory protein, additional roles for Tat in HIV replication have been described, including reverse transcription. Tat achieves function through direct interaction with viral proteins, including reverse transcriptase, and numerous cellular proteins including cyclin T1, RNA polymerase 11, protein kinase R (PKR), p300/CBP, and P/CAF. Despite our advanced knowledge of how Tat operates, this has not yet resulted in the discovery of effective agents capable of targeting various Tat functions. Nevertheless, Tat remains an attractive, virus-specific molecule and detailed understanding of specific protein interaction holds promise for future drug discovery.

Identification and Characterization of New Small Molecule Inhibitors of Picornavirus Replication

The Picornaviridae family consists of positive-strand RNA viruses that are the causative agents of a variety of diseases in humans and animals. Few drugs targeting picornaviruses are available, making the discovery of new antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. The antiviral effect of these compounds is not likely related to their known cellular targets because other inhibitors targeting the same pathways did not inhibit viral replication. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited the formation of a functional replication complex, while E5(1) and E7(2) affected replication after the replication complex had formed. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Poliovirus resistant to E7(2) had a single mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target either PI4KIIIβ (major enviroxime-like compounds) or OSBP (minor enviroxime-like compounds), cellular factors involved in lipid metabolism and shown to be important for replication of diverse positive-strand RNA viruses. We classified E7(2) as a minor enviroxime-like compound, because the localization of OSBP changed in the presence of this inhibitor. Interestingly, both E7(2) and major enviroxime-like compound GW5074 interfered with the viral polyprotein processing. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. Studies with these compounds shed light on pathways shared by diverse picornaviruses that could be potential targets for the development of broad-spectrum antiviral drugs.

MECHANISM OF UPREGULATION OF PHOSPHATIDYLCHOLINE SYNTHESIS DURING PICORNAVIRUS INFECTION AND ITS ROLE IN THE DEVELOPMENT OF VIRAL REPLICATION ORGANELLES

Picornaviruses are a group of human and animal pathogens capable of inflicting serious public health diseases and economic burdens. Treatments options through vaccines for prevention or antivirals to cure infection are not available for the vast majority of these viruses. These shortcomings, in the development of vaccines or antivirals therapeutic, are linked to the genetic diversity and to an incomplete understanding of the biology of these viruses. Despite the diverse host range, this group of positive-strand RNA viruses shares the same replication mechanisms, including the development of membranous structures (replication organelles) in the cytoplasm of infected cells. The development of these membranous structures, which serve as sites for the replication of the viral RNA genome, has been linked to the hijacking of elements of the cellular membrane metabolism pathways. Here we show that upon picornavirus infection, there is a specific activation of acyl-CoA synthetase enzymes resulting in strong import and accumulation of long chain fatty acids in the cytoplasm of infected cells. We show that the newly imported fatty acids serve as a substrate for the upregulation of phosphatidylcholine synthesis required for the structural development of replication organelles. In this work, we identified that acyl-CoA synthetase long chain 3 (ACSL3) is required for the upregulation of lipids syntheses and the replication of poliovirus. We have shown that the poliovirus protein 2A was required but not sufficient for the activation of import of long chain fatty acids in infected cells. We demonstrated that the fatty acid import is upregulated upon infection by diverse picornaviruses and that such upregulation is not dependent on activation of ER stress response or the autophagy pathways. In this work, we have demonstrated that phosphatidylcholine was required for the structural development of replication organelles. Phosphatidylcholine synthesis was dispensable for the production of infectious particles at high MOI but required at a low MOI for the protection of the replication complexes from the cellular innate immunity mechanisms.

Correlation Between Testosterone And Psa Kinetics In Metastatic Prostate Cancer Patients Treated With Diverse Chemical Castrations.

To assess total testosterone and prostatic-specific antigen (PSA) kinetics among diverse chemical castrations, advanced-stage prostate cancer patients were randomized into three groups of 20: Group 1, Leuprolide 3.75 mg; Group 2, Leuprolide 7.5 mg; and Group 3, Goserelin 3.6 mg. All groups were treated with monthly application of the respective drugs. The patients' levels of serum total testosterone and PSA were evaluated at two time periods: before the treatment and 3 months after the treatment. Spearman's rank correlation coefficient was utilized to verify the hypothesis of linear correlation between total testosterone and PSA levels. At the beginning the patients' age, stage, grade, PSA, and total testosterone were similar within the three groups, with median age 72, 70, and 70 years in Groups 1, 2, and 3, respectively. Three months after the treatment, patients who received Leuprolide 7.5 mg presented significantly lower median total testosterone levels compared with Goserelin 3.6 mg and Leuprolide 3.75 mg (9.5 ng/dL vs. 20.0 ng/dL vs. 30.0 ng/dL, respectively; p = .0072), while those who received Goserelin 3.6 mg presented significantly lower PSA levels compared with Leuprolide 7.5 mg and Leuprolide 3.75 mg (0.67 vs. 1.86 vs. 2.57, respectively; p = .0067). There was no linear correlation between total testosterone and PSA levels. Overall, regarding castration levels of total testosterone, 28.77% of patients did not obtain levels ≤50 ng/dL and 47.80% did not obtain levels ≤20 ng/dL. There was no correlation between total testosterone and PSA kinetics and no equivalence among different pharmacological castrations.

Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. Methodology and Principal Findings: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. Conclusion and Significance: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, ""promiscuous"" ( multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II-transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

Genetically Diverse Coronaviruses in Captive Bird Populations in a Brazilian Zoological Park

This study aimed to investigate the occurrence of coronaviruses (CoVs) in captive birds placed inside a zoological park in Brazil. The role of captive birds in the epidemiology of CoVs in the tropics is poorly understood. A total of 25 (n = 25) different species were tested for viral RNA using individual fecal samples collected from healthy birds. Reverse transcription-polymerase chain reaction targeting the 30 untranslated region was used to detect CoV RNA, and positive samples were submitted for sequence analysis. The phylogenetic search revealed nine mutations in the black shouldered peafowl (Pavus cristatus) CoV sequence, which clustered separately from samples previously described in England. This is the first report on the detection of the CoV genome in captive birds in Brazil.

Do plant species influence soil CO(2) and N(2)O fluxes in a diverse tropical forest?

To test whether plant species influence greenhouse gas production in diverse ecosystems, we measured wet season soil CO(2) and N(2)O fluxes close to similar to 300 large (>35 cm in diameter at breast height (DBH)) trees of 15 species at three clay-rich forest sites in central Amazonia. We found that soil CO(2) fluxes were 38% higher near large trees than at control sites >10 m away from any tree (P < 0.0001). After adjusting for large tree presence, a multiple linear regression of soil temperature, bulk density, and liana DBH explained 19% of remaining CO(2) flux variability. Soil N(2)O fluxes adjacent to Caryocar villosum, Lecythis lurida, Schefflera morototoni, and Manilkara huberi were 84%-196% greater than Erisma uncinatum and Vochysia maxima, both Vochysiaceae. Tree species identity was the most important explanatory factor for N(2)O fluxes, accounting for more than twice the N(2)O flux variability as all other factors combined. Two observations suggest a mechanism for this finding: (1) sugar addition increased N(2)O fluxes near C. villosum twice as much (P < 0.05) as near Vochysiaceae and (2) species mean N(2)O fluxes were strongly negatively correlated with tree growth rate (P = 0.002). These observations imply that through enhanced belowground carbon allocation liana and tree species can stimulate soil CO(2) and N(2)O fluxes (by enhancing denitrification when carbon limits microbial metabolism). Alternatively, low N(2)O fluxes potentially result from strong competition of tree species with microbes for nutrients. Species-specific patterns in CO(2) and N(2)O fluxes demonstrate that plant species can influence soil biogeochemical processes in a diverse tropical forest.

Wolbachia replication and host cell division in Aedes albopictus

Wolbachia pipientis is an obligate intracellular endosymbiont of a range of arthropod species. The microbe is best known for its manipulations of host reproduction that include inducing cytoplasmic incompatibility, parthenogenesis, feminization, and male-killing. Like other vertically transmitted intracellular symbionts, Wolbachiarsquos replication rate must not outpace that of its host cells if it is to remain benign. The mosquito Aedes albopictus is naturally infected both singly and doubly with different strains of Wolbachia pipientis. During diapause in mosquito eggs, no host cell division is believed to occur. Further development is triggered only by subsequent exposure of the egg to water. This study uses diapause in Wolbachia-infected Aedes albopictus eggs to determine whether symbiont replication slows or stops when host cell division ceases or whether it continues at a low but constant rate. We have shown that Wolbachia densities in eggs are greatest during embryonation and then decline throughout diapause, suggesting that Wolbachia replication is dependent on host cell replication.

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm(2) embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T-1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).