Kyasanur Forest disease virus (KFDV) of the Flaviviridae virus family has caused seasonal infections and periodic outbreaks in Karnataka, India. First identified in 1957, KFDV annually infects 400-500 people and has a fatality rate of 3-5%; there are no approved antivirals and the existing licensed vaccine’s effectiveness appears to be questionable. Many tools for KFDV research are limited and this work sought to develop methods for analysing antivirals, including interferon (IFN)-α/β species. The BHK-21 (ATCC) cell line allowed for high virus propagation and distinguishable cytopathic effects (CPE) for determining antiviral effectiveness. The additional tool of a reverse genetics system expressing a full-length cDNA KFDV genome with a GFP reporter failed to propagate, despite numerous GFP genome-insertion strategies. The clinically approved IFN-α2a or IFN-α2b has had variable success at combatting flavivirus diseases in people, especially in the immuno-compromised. The continued passaging of KFDV-infected cells with repeated IFN-α2a treatment did not eliminate KFDV and had little effect on infectious particle production. IFN-αspecies, αWA and α were more effective than IFN-α2a and α2b at reducing KFDV; however dose ranges indicated that while low concentrations could limit CPE, higher concentrations were needed to inhibit virion release. Avoidance of IFN-α/β through Jak/STAT signalling repression was attributed to the NS5 protein, specifically the RdRp domain based on data obtained with luciferase and vesicular stomatitis virus (VSV) recovery assays. However, the mechanism appears to act subsequently to STAT1/2 activation without NS5 binding to any Jak/STAT components. A non-infectious, replicative system serving as a platform for antiviral drug testing against KFDV in a high throughput manner could only provide luciferase signals when the NS proteins capable of driving replication, were supplied in cis (subgenomic) but not in trans (antigenome). To conclude, IFN-α species such as IFN-αWA may be better suited than the licensed IFN-α2a for treatment of KFDV infections; however, IFN effects appear to be subdued in vitro due to the actions of the NS5 protein. While IFN may not be a successful antiviral against KFDV, the work in this thesis provides a foundation for evaluating other potential anti-KFDV therapeutics. / February 2016

Typhoid fever remains an important public health problem globally. Cluster randomized effectiveness trials with typhoid Vi polysaccharide vaccine were conducted in Kolkata, India and Karachi, Pakistan, to provide evidence for vaccine introduction. While efficacy trials are limited to estimate vaccine's performance on the vaccine recipients, effectiveness trials consider the public health impact, notably the herd protection, or indirect effect, which can only be seen when vaccines are administered to groups rather than to individuals. The observed total protection by the Vi polysaccharide vaccine in school-aged children was consistent in Kolkata and Karachi (61% and 56%, respectively), and was associated with minimal side-effects. The total protection in young children, however, was different (80% in Kolkata and no protection in Karachi). The Kolkata trial demonstrated significant herd protective effects, as demonstrated by indirect protection of non-vaccinees (45%), which was not shown in the Karachi trial. The difference in the effectiveness estimates between the trials may be due to the difference in study design and the population characteristics. Immunogenicity studies were undertaken for randomly selected persons from both sites at pre-vaccination, 6 weeks, and 2 years post-vaccination. Serum Vi antibody titres (IgG) were measured through ELISA. At baseline, the GMTs were below the protective level for both sites. At six weeks after vaccination, though there is a significant increase in the GMTs in children from both site, the level of GMTs were significantly lower from those in Karachi (2,307.0 ELU vs. 1,189.1 ELU). GMT declined from 6 week to 2 year testing points for both sites but maintained the protective level. These effectiveness trials gave a conclusive evidence of the protection conferred by the Vi polysaccharide vaccine in children older than 5 years of age. Targeted vaccination programme in high endemic areas, as stipulated in the WHO Position Paper, suggest the potential for effective control of typhoid fever in places like India and Pakistan with the school-based Vi vaccination.

The disease vectors Aedes aegypti and Aedes albopictus are serious and growing threats to global health. As vectors of the arboviruses dengue fever and chikungunya, these mosquitoes are responsible for hundreds of millions of cases and thousands of deaths each year. Absent specific treatments or vaccines, effective control of mosquito populations remains the only option for tackling a growing public health challenge. More effective control tools are urgently needed. Recently, a novel approach to pest population control has been developed based on the release of insects carrying a repressible, dominant lethal allele. This approach has achieved dramatic reductions in Ae. aegypti populations in regulated open field experiments. Despite this success, there remains scope to improve upon the current technology. It is proposed that an 'ideal' strain would combine the following features: (i) repressible lethality in late juvenile phases; (ii) a mechanism for removing females at an early developmental stage in the release generation; and (iii) orthogonal expression control mechanisms allowing both these systems to be combined in a single strain. This thesis describes research undertaken in pursuit of a 'next generation' strain. Two novel promoters from putative Osiris genes have been identified which confer a 10²-10³ – fold up-regulation in transgene expression specific to late pupal stages. One of these 'Osiris' promoters has been used to develop transgenic Aedes aegypti strains. 5 lines showed pupal-specific lethality of 98-100% penetrance, which was repressed in the presence of tetracycline. An Ae. albopictus orthologue of the sex-determining gene doublesex (dsx) has been isolated and characterised and a female-specific expression system developed. Transgenic lines show female-specific expression of a transgene; however, there remains some 'leaky' expression in male insects. Finally, a potential expression control tool based on an auxin-inducible expression system has been investigated. 11 different transgenic lines were developed based on three different construct designs. None showed auxin-inducible expression of a transgene.

En Afrique, les fièvres récurrentes à Borrelia sont des pathologies négligées transmises par les arthropodes et responsables de septicémie mortelle et d'autres manifestations cliniques, en particulier d'avortement chez les femmes enceintes. Quatre Borrelia différentes sont actuellement cultivées de prélèvements cliniques et de vecteurs, il s'agit de Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. Ces différentes espèces ont été initialement séparées les unes des autres sur la base de leur répartition géographique et de leur vecteur. Au cours de ce travail de thèse, nous avons réalisé le séquençage et l'annotation du génome de Borrelia crocidurae. Ceci nous a permis de comparer le génome de trois espèces séquencées: Borrelia crocidurae, Borrelia duttonii et Borrelia recurrentis. Cette comparaison indique que ces trois espèces sont extrêmement proches comme cela avait déjà été montré par la comparaison de la séquence du gène 16S ARN ribosomal de chacune de ces espèces, montrant une similarité comprise entre 99,7 et 99,9%. L'analyse de données génomiques permet de conforter les résultats antérieurs basés sur l'analyse de quelques gènes et de proposer que ces trois espèces ne forment qu'une seule espèce génomique présentant trois écotypes avec une relative spécificité de vecteur, de répartition géographique et d'évolution clinique. La très grande similitude entre Borrelia crocidurae, Borrelia duttonii et Borrelia recurrentis constitue un obstacle pour la mise au point de techniques de diagnostic direct de ces espèces dans les prélèvements de vecteur ou dans les prélèvements humains. / In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia. Borrelia crocidurae, Borrelia duttonii, Borrelia hispanica and Borrelia recurrentis are the currently cultured causative agents in Africa. The relapsing fever borreliae species were initially distinguished one from another on the basis of geography and vector. we performed the genome sequencing of Borrelia crocidurae. At the genomic level the four species are highly similar as illustrated by the 16S rRNA gene sequence of each species, resulting in an overall high sequence similarity of 99.7 to 99.9% between the four species. Genomic analyses of Borrelia crocidurae, Borrelia duttonii and Borrelia recurrentis further indicated that they in fact forming an unique bacterial species, each one of the three species could be regarded as an ecotype of an unique species with preferential arthropod vector, geographic distribution and clinical outcome, rather than an unique bacterial species. The high similarity between species remained an obstacle for the diagnosis at the species level. Currently the identification of relapsing fever borreliae relies upon a few phenotypic traits and the detection of single nucleotide polymorphisms in the 16S rRNA and flabB, glpQ genes and the 16S-23S ribosomal RNA intergenic spacer (IGS). In this study, based on comparative genomic anaylsis between the published genomic sequence and partial sequence in the genbank, we developed multiplex, quantitative real-time PCR detecting any relapsing fever Borrelia and specifically B. crocidurae, B. hispanica and B. duttonii/B. recurrentis.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Manuel Borca / Stephen Higgs / African swine fever (ASF) is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus. ASFV is endemic in Sardinia and Saharan Africa and has been recently expanded from the Caucasus to Eastern Europe. There is no vaccine to prevent the disease and current control measures are limited to culling and restricted animal movement. Swine infected with attenuated strains are protected against challenge with a homologous virulent virus, but there is limited knowledge of the host immune mechanisms generating that protection. Swine infected with Pret4 virus develop a fatal severe disease, while a derivative strain lacking virulence-associated gene 9GL (Pret4Δ9GL virus) is completely attenuated. Swine infected with Pret4 Δ9GL virus and challenged with the virulent parental virus at 7, 10, 14, 21, and 28 dpi showed a progressive acquisition of protection (from 40% at 7 dpi to 80% at 21 and 28 dpi). This animal model was used to associate the presence of host immune response and protection against the challenge. Anti-ASFV antibodies and cytokines in serum, as well as ASFV-specific IFN-γ production in PBMCs, were assessed in each group. Interestingly, with the exception of ASFV-specific antibodies in the surviving swine challenged at 21 and 28 dpi, no solid association between any of the parameters assessed and the extent of protection could be established. These results were corroborated using a similar model based on the use of a rationally attenuated derivative of the highly virulent strain Georgia 2007. These results, encompassing data from 114 immunized swine, underscore the complexity of the system under study where it is very plausible that protection against disease or infection relies heavily on the concurrence and or interaction of different host immune mechanisms.

Albert Baldwin Wood and his screw pumps modernized New Orleans by bringing flood-free streets and cleaner water to the city while providing the potential for growth by pumping swamp lands dry. While Wood was never part of the local Progressive movement, his work with the pumps fit with Progressive initiatives for modernization. At first, the screw pumps removed rain water from the streets. Then New Orleans expanded the drainage to include sewerage removal and water purification. The pumps successfully drained thousands of acres of land once considered uninhabitable swamp land. This additional land extended New Orleans city limits but also aided in the acceleration of residential segregation. Cities from around the world used the designs for the screw pump and consulted Wood for advice on drainage systems.

A retired actress and her quirky family trap four guests in elaborately woven games in Noel Coward’s Hay Fever. Within the concept of “playing house,” the glamour of Coward’s work lives on through scenery, costumes, and lighting, while his quirkier side is highlighted in hopes of making his Comedy of Manners accessible to a new audience.

Les macrophages sont fonctionnellement polarisés en macrophages inflammatoires et microbicides (M1) ou immunorégulateurs (M2). Que les monocytes circulants soient polarisables n’est pas démontré. Nous avons étudié la signature transcriptionnelle par microarray et RT-PCR en temps réel de monocytes humains stimulés par l’IFN-γ, un inducteur des macrophages M1 et l’IL-4, un inducteur des macrophages M2. Leur profil de réponse précoce est dépendent de l’agoniste alors que la réponse tardive des monocytes est similaire qu’ils soient stimulés par l’IFN-γ ou l’IL-4. Cette approche dynamique de la réponse monocytaire permet probablement une étude bien plus pertinente des patients atteints d’une fièvre Q que le modèle de polarisation macrophagique. Par ailleurs, la prévalence de la fièvre Q est plus importante chez l’homme que chez la femme. Comme il a été montré que des gènes associés au cycle circadien sont modulés chez les souris infectées par Coxiella burnetii, la bactérie responsable de la fièvre Q, nous avons étudié ces gènes au cours de la fièvre Q. C’est ainsi que le gène Per2 est fortement exprimé chez les hommes atteints d’une fièvre Q aiguë. Ces résultats suggèrent donc que la modulation de gènes circadiens est associée à une maladie infectieuse chez l’homme. L’expression des gènes LNX1 and LNX2, qui codent deux enzymes impliquées dans le catabolisme des protéines, est accrue dans l’endocardite de la fièvre Q mais pas dans la fièvre Q aiguë. / Macrophages are functionally polarized into inflammatory and microbicidal (M1) and immunoregulatory (M2) cells. If circulating monocytes may be polarized is not known. We determined the transcriptional signatures of human monocytes stimulated with IFN-γ and IL-4, known to induce the polarization of macrophages into M1 and M2 cells, respectively, using microarrays and real-time RT-PCR. We found that monocytes exhibited an early pattern of activation specific to IFN-γ or IL-4 and a late pattern of activation common to both agonists. The selected biomarkers of early and late responses were tested in patients with Q fever. We showed that the kinetic model of monocyte activation enables a dynamic approach for the evaluation of patients with acute Q fever or Q fever endocarditis. On the other hand, it is known that the prevalence of Q fever is related to sex and is higher in men than in women. Based on previous studies on an experimental model of infection by Coxiella burnetii, the agent of Q fever, we hypothesized that circadian genes are differently modulated in men and women with Q fever. We showed that the expression of the Per2 gene was significantly increased in males with acute Q fever compared with healthy volunteers but did not differ in females with Q fever and healthy females. These results suggest that that the modulation of circadian genes is associated with a human infectious disease. We also found that the expression of LNX1 and LNX2 genes that encode two enzymes involved in protein degradation is increased in Q fever endocarditis but not in acute Q fever.

No description available.

Orientador: Marcos Rogério André / Coorientador: José Maurício Barbanti Duarte / Banca: Karina Paes Bürger / Banca: Ana Patrícia Yatsuda Natsui / Resumo: Coxiella burnetii é uma bactéria Gram-negativa intracelular obrigatória, que além de considerado agente zoonótico causador da Febre Q em vários países do mundo, foi classificado como um potencial agente de bioterrorismo. Bovinos, ovinos e caprinos representam as fontes de infecção mais frequentemente associadas à ocorrência da enfermidade em humanos, entretanto animais selvagens também podem atuar como importantes fontes de infecção. Desta forma, o presente estudo tem como objetivo investigar a ocorrência de Coxiella burnetii em ruminantes domésticos e selvagens no Brasil. Para tal, 188 amostras de sangue de cervídeos (143 Blastocerus dichotomus, 11 Ozotocerus bezoarticus, 27 Mazama gouazobira, 4 M. bororo e 3 M. americanum), capturados nos estados de MS, SP e MG, foram submetidas à extração de DNA e, subsequentemente, à nested (n)PCR para C. burnetii baseada no elemento de inserção repetitivo IS1111 do gene heat shock protein (htpAB). Além disso, 169 amostras de soro de cervídeos foram submetidas à Reação de Imunofluorescência para detecção de anticorpos IgG anti-C. burnetii. Amostras de soros de bovinos apresentando desordens reprodutivas foram submetidas às Reações de Vírus Neutralização para BoHV-1 e BVD, Soroaglutinação Microscópica para Leptospira spp., Reação de Imunofluorescência Indireta for C. burnetii e Toxoplasma gondii, e Ensaio de Imunoabsorção Enzimática Indireto para Neospora caninum e Trypanosoma vivax. Todas as amostras de sangue mostraram-se negativas na nP... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Coxiella burnetii is an obligate intracellular Gram-negative bacterium, which, in addition to being considered a zoonotic agent that causes Q fever in several countries of the world, has been classified as a potential bioterrorism agent. Cattle, sheep and goats represent the most frequent sources of infection associated with the occurrence of the disease in humans, however, wild animals can also act as important sources of infection. In this way, the present study aims to investigate the occurrence of Coxiella burnetii in domestic and wild ruminants in Brazil. To this end, 188 cervus blood samples (143 Blastocerus dichotomus, 11 Ozotocerus bezoarticus, 27 Mazama gouazobira, 4 M. bororo and 3 M. americanum), captured in the states of MS, SP and MG, were subjected to DNA extraction and, subsequently to the nested (n) PCR for C. burnetii based on the heat shock protein (htpAB) gene IS1111 insertion element. In addition, 169 cervical serum samples were submitted to Immunofluorescence Reaction for the detection of anti-C. burnetii IgG antibodies. Adicionaly, samples of bovine sera presenting reproductive disorders were submitted to the Virus Reaction Neutralization for BoHV-1 and BVD, Microscopic Soroagglutination for Leptospira spp., Indirect Immunofluorescence Reaction for C. burnetii and T. gondii, and Indirect Enzyme Immunoabsorption Assay for N caninum and T. vivax. All blood samples were negative in nPCR, evidencing absence of circulating DNA of C. burnetii in the sampled c... (Complete abstract click electronic access below) / Mestre