Tesi sul tema "Resolution of infection"

Herpes simplex virus (HSV) modulates the host cell’s proteome and transcriptome during infection to fulfil the needs of the virus for productive replication and transmission. Using bioorthogonal precursors and click chemistry, I have examined spatiotemporal aspects of global protein synthesis during single step replication and cell-to-cell transmission with results revealing new insights into the complex spatial interplay between translational control processes, protein localisation and transcription during HSV infection. For the first time, translational suppression and recovery is visualised at the single cell level, reflecting a very early biphasic switch in translational control. The biphasic switch is dependent on the RNase activity of HSV virion host shutoff protein (vhs), and vhs also mediates eIF4H nuclear translocation coupled to the initial suppression. During translational recovery, my results also show rapid accumulation of newly synthesised proteins in novel subnuclear domains termed newly synthesised protein domains (NPDs). ICP22 is specifically required for NPD formation and shows selective recruitment to these domains. Additionally, a host protein, SSRP1, is also displaced from its normal localisation in the nucleolus and selectively recruited to NPDs very early in infection, dependent on ICP22 expression. Furthermore, spatial analysis of newly transcribed RNA also reveals that early after infection transcripts accumulate in irregular structures termed RNA islands which form distinct populations. While a fraction of RNA islands localises juxtaposed with a subpopulation of NPDs, the majority of this newly synthesised RNA shows precise colocalisation in distinct domains which also recruits a cellular RNA helicase, p68. These later RNA domains adjoin foci containing the immediate-early transcriptional regulator ICP4 in a specifically organised manner. In addition to profound qualitative changes observed during translational recovery, quantitative proteomics and identification of the newly synthesised nuclear proteome using HPG pulsed-SILAC with LC- MS/MS indicates coordinated mobilisation of distinct cellular pathways, in particular eIF2 signalling pathway and RNA post-transcriptional modification. These results reveal new features of protein metabolism during infection, and involvement of ICP22 in newly synthesised protein processing pathways.

Pseudomonas aeruginosa (P.a) est un bacille Gram négatif responsable d’infections chroniques associées à une mortalité élevée due à la prédilection de la bactérie à développer une résistance aux antibiotiques et l’inefficacité des thérapies actuelles. Notre groupe a montré dans un modèle d’infection aigue chez la souris, que l’élastase B (LasB), un facteur de virulence de P.a, dégrade la cytokine IL-6 et la molécule antimicrobienne Elafine et que la surexpression de ces deux médiateurs confère une protection aux souris en diminuant l’inflammation et augmentant la réparation. Les macrophages alvéolaires représentent la population myéloïde la plus abondante dans l’espace alvéolaire et jouent un rôle clé dans le maintien de l’homéostasie, l’initiation & la résolution de l’inflammation. Compte tenu de leur importance, ils sont très étudiés dans le cadre de développement de nouvelles approches de thérapie cellulaire. Nous avons donc émis l’hypothèse que le macrophage alvéolaire qui est également cible de P.a et de LasB plus particulièrement, puisse être un outil adéquat pour le transfert de la protection IL-6- et Elafine-médiée. L’objectif principal de ce travail est de modifier le macrophage avec des vecteurs adénoviraux permettant la surexpression de l’IL-6 et de l’Elafine, et de l’utiliser comme un outil thérapeutique dans un modèle de transplantation intrapulmonaire suivie d’une infection par P.a. Nous montrons que le transfert de macrophages génétiquement modifiés avec l’IL-6 et l’Elafine est protecteur. L’Elafine induit dans le macrophage une signature IL6/IL10/peptides antimicrobiens qui, en synergie avec l’IL-6, confère un phénotype régulateur à l’unité alvéolaire
Pseudomonas aeruginosa (P.a) is a Gram-negative bacillus responsible for chronic infections associated with high mortality due to the bacterium's predilection for developing antibiotic resistance and the inefficacy of current therapies. Our group showed in a model of acute infection in mice that Elastase B (LasB), a virulence factor of Pa, degrades the cytokine IL-6 and the antimicrobial molecule Elafine and that the overexpression of these two mediators provides protection to mice by decreasing inflammation and increasing repair. Alveolar macrophages represent the most abundant myeloid population in the alveolar space and play a key role in maintaining homeostasis, initiation and resolution of inflammation. Given their importance, they are very much studied in the development of new approaches to cell therapy. We therefore hypothesized that the alveolar macrophage which is also targeted by P.a and LasB more particularly, may be an adequate tool for the transfer of IL-6- and Elafine-mediated protection. The main objective of this work is to modify the macrophage with adenoviral vectors allowing the overexpression of IL-6 and Elafine, and to use it as a therapeutic tool in an intrapulmonary transplantation model followed by a Pa infection We show that the transfer of genetically modified macrophages with IL-6 and Elafine is protective. Elafine induces in the macrophage an IL6 / IL10 / antimicrobial peptide signature which, in synergy with IL-6, confers a regulatory phenotype to the alveolar unit

Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.

Intensive care units (ICU) and Surgical Center are noteworthy environments, because patients with poor health that frequent these places are subjected to acquire infections from the air of these environments. The aim of this study was to evaluate the air quality during the dry and rainy seasons of air-conditioned rooms of the NICU, neonatal intensive care unit, (A, B and C) , surgical center and exterior area at the University Hospital HUPAA/UFAL, Maceió-AL, for parameters recommended by ANVISA, and identify the mycoflora found and quantify the bacteria in the air. A number of 22 points of collects were performed, totaling 220 samples of air taken through the methodology specified by ANVISA s No. 9 Resolution, which consists in the definition of the parameters of bioaerosol, CO2, temperature, relative humidity, air velocity and aerialdispersoids. Macro and microscopic features were compared in order to identify the mycoflora and yeasts were identified by PCR with species-specific primers. From the 3.839 fungal colony forming units (CFU) isolated, 1.085 CFU were identified as belonging to 21 genera distributed among 63 species. Mycelia Sterilia with 121 CFU (11.1%) and Cladosporium cladosporioides with 78 CFU (7.2%) were the most frequent species in rainy season. Among the yeast species, Candida parapsilosis and C. tropicalis had the biggest frequency, both with four isolates corresponding to 4.7% each. The means were statistically significant for the parameters CO2, temperature, humidity and aerialdispersoids, not being significant for the air speed parameter. The observed values shows that the environments tested in the hospital were out of compliance for most of the parameters recommended by health surveillance in relation to the indoor air quality. Corrective measures are needed to remedy the possible sources of contamination in hospitals in order to comply with the four technical standards recommended by the National Agency for Sanitary Vigilance, contributing to the reduction of the levels of hospital infection, and the number of deaths related to it.
Fundação de Amparo a Pesquisa do Estado de Alagoas
As UTI s e o centro cirúrgico são ambientes hospitalares que merecem destaque, pois os pacientes nestes locais estão sujeitos a adquirir infecções provenientes do ar desses ambientes por apresentarem a saúde debilitada. O objetivo deste trabalho foi avaliar a qualidade do ar durante os períodos seco e chuvoso dos ambientes internos climatizados A, B e C da UTN, do centro cirúrgico e do ponto externo do Hospital Universitário HUPAA/UFAL, Maceió-AL, com relação aos parâmetros recomendados pela ANVISA, além de identificar a microbiota fúngica e quantificar as bactérias presentes no ar. Foram realizadas 22 coletas, totalizando 220 amostragens de ar realizadas através da metodologia indicada pela Resolução nº 9 da ANVISA que consiste na determinação dos parâmetros de bioaerossóis, CO2, temperatura, umidade relativa, velocidade do ar e aerodispersóides. Para identificação dos fungos filamentosos foram comparadas as características macro e microscópicas e as leveduras foram identificadas através da técnica de PCR com iniciadores espécie-específicos. Das 3.839 UFC fúngicas isoladas foram identificadas 1.085 UFC pertencentes a 21 gêneros distribuídos em 63 espécies, sendo Mycelia Sterilia com 121 (11,1%) UFC e Cladosporium cladosporioides com 78 (7,2%) UFC as espécies mais freqüentes no período chuvoso. Entre as leveduras, Candida parapsilosis e C. tropicalis foram as mais ocorrentes, ambas com quatro isolados correspondendo a 4,7% cada. As médias foram estatisticamente significativas para os parâmetros CO2, temperatura, umidade relativa, aerodispersóides e bioaerossóis, não sendo significativas para o parâmetro velocidade do ar. Os valores observados demonstram que os ambientes hospitalares estudados estavam fora de conformidade para maioria dos parâmetros recomendados pela vigilância sanitária em relação à qualidade do ar de interiores. Medidas corretivas são necessárias para remediar as possíveis fontes de contaminação nas unidades hospitalares de forma a atender as quatro normas técnicas preconizadas pela Agência Nacional de Vigilância Sanitária, contribuindo para minimizar os níveis de infecção hospitalar e o número de óbitos a esta relacionados.

The eco-epidemiology of bovine tuberculosis (Tb) in wild deer (mainly red deer Cervus elaphus) in New Zealand was investigated. Bovine Tb is caused by Mycobacterium bovis. Specific aims were to clarify the likely routes of infection in deer, and to determine the status of deer as hosts of Tb, the likely rates and routes of inter- and intra-species transmission between deer and other wildlife hosts, the role of deer in spreading Tb, and the likely utility of deer as sentinels of Tb presence in wildlife. As the possum (Trichosurus vulpecula) is the main wildlife host of Tb, the research also included some investigation of transmission routes in possums. Patterns of infection were measured in 994 deer killed between 1993 and 2003. Tb prevalence varied between areas (range 8–36%). Few deer had generalised infection, with 21–68% of infected deer having no visible lesions, depending on the area. The retropharyngeal lymph nodes and oropharyngeal tonsils were commonly infected. No dependent fawns less than 0.75 years old were infected, indicating intra-species transmission is rare in wild deer. Where possums were not controlled, the net (cumulative) force of infection in young (1–4 y) deer was 0.10–0.24 per year in males and 0.09–0.12 per year in females, but much lower in older deer (less than 0.05 per year). Possum control reduced the net force of infection quickly, and eventually to zero. However, Tb persisted in possum-controlled areas through immigration of infected deer and, for almost a decade, through the survival of resident deer infected before possum control. Tb was lost from infected deer at an exponential rate of 0.13 per year, mostly as a result of deer recovering from infection rather than dying from it. Wild deer do die of Tb, but there was no discernible effect on age structure. The occurrence of infection in deer was not linked to the local deer or possum density at their kill sites (i.e. in their home range), but the area-wide prevalence of Tb in deer was closely correlated with Tb levels in possums, which were in turn correlated with area-wide measures of possum density. For wild deer in New Zealand, Tb is a persistent but usually inconsequential disease of the lymphatic system. It is acquired mainly by young independent deer, usually orally via the tonsils, and probably as a result of licking infected possums. Many species fed on deer carrion, including possums. Most possums encountering carrion did not feed on it, but a few fed for long periods. Other scavengers such ferrets (Mustela furo), hawks (Circus approximans), and weka (a hen-sized flightless native bird; Gallirallus australis) fed in a way that probably increased the infectivity of carrion to possums. Commercial deer hunting may have facilitated the historical establishment of Tb in possums. Scavenging (including cannibalism) and interactions with dead and dying possums are identified for the first time as potentially important routes for transmission of Tb to possums, and I develop new hypotheses involving peri- and post-mortem transmission in possums that explain many of the epidemiological patterns that are characteristic of the disease in possum. In continuous native forest, deer home range size averaged 250 hectares for six young females, and over twice that for two males. Over 90% of infected deer are likely to die within 2 km (females) or 6 km (males) of where they acquired Tb, but deer could occasionally carry Tb up to 30 km. Deer will be useful as sentinels, but only where other sentinels are rare, because the force of infection for a deer with a single infected possum in its home range is only 0.004 per year, compared to greater than 0.2 per year for deliberately released pigs. Deer are occasionally capable of initiating new cycles of infection in wildlife, but deer control is not essential to eradicate Tb from wildlife.

Innate immune activation is thought to play a central role in IBD pathogenesis because genetic polymorphisms in NOD2 and NLRP3, cytosolic innate immune receptors belonging to the NLR family, have been associated with IBD susceptibility. However, the mechanisms through which NLR mutations predispose to IBD remain unclear. The aim of this project was to dissect the functional roles of different NLRs in intestinal inflammation. Using the well-established DSS-induced colitis model as well as experimental models of IBD based on infection with Helicobacter hepaticus, we found that Nod2 expression was significantly increased at the peak of intestinal inflammation, and remained elevated throughout the resolution process. This observation suggests a possible role for Nod2 in the resolution of inflammation. Conversely, upon infection with the acute intestinal pathogen Citrobacter rodentium, Nlrp3-/- mice suffered from increased bacterial colonization as early as 3 days post infection, resulting in exacerbated intestinal inflammation and severe weight loss. Analysis of irradiation bone marrow chimeras showed that the protection required Nlrp3 activation in the non-haematopoietic compartment. Furthermore, this protective mechanism was independent of the inflammasome-associated cytokines IL-1β or IL-18. Therefore, this study implicates Nlrp3 activation in intestinal tissue cells as having a crucial role in controlling pathogenic bacterial colonization, providing a potential mechanism by which NLRP3 polymorphisms could lead to increased susceptibility to IBD.

Background: The diagnosis of bloodstream infection is a significant challenge for healthcare providers and is often associated with severe illness (sepsis) and poor outcomes. Rapid detection and identification of pathogens followed by characterisation of antibiotic resistance could help direct early treatment and improve patient care. Standard blood culture methods, which usually take 2-5 days to complete, can confirm if there is a bacteraemia or not in suspected patients. However, molecular approaches have been developed and are being increasingly investigated to overcome disadvantages of culture. One of the main potentials of molecular techniques is that they should be able to identify pathogens within a short time which could help clinicians treat patients earlier with rational antimicrobial therapy and limit overuse of antibiotic exposure. Objectives: To present the development and optimisation of a simple, rapid and cost-effective Real Time PCR methods combined with a High Resolution Melting Analysis (HRMA) approach, to detect and identify common bacteria associated with bloodstream infections. Approach: 16S rRNA and Gram classification primers were used on a broad range real-time PCR for molecular Gram typing and HRMA in a single run. Differentiation of bacterial species was achieved using a multi-parameter, decision-tree approach based on Gram typing, grouping according to melting temperature (Tm) and sequential comparisons of melting profiles (Curve shapes) against reference organisms. Findings: A preliminary validation was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. 50 isolates contained organisms present on the panel and 96% of these were identified correctly at genus or species level. A correct Gram classification was reported for all 53 isolates. The strategy of amplification of the bacterial signal to an appropriate level using a short term pre-culture system (STPCS) for up to 12 hours prior to HRMA analysis significantly improved the overall sensitivity of the assay in spiked blood. Conclusion: This study suggests that a PCR-HRMA approach could be used as an alternative cheap approach to other molecular approaches for rapid detection and identification of bacteria responsible for >95% of bloodstream infections especially when combined with a Short Term Pre-Culture System (STPCS). Such development together with the current standard culture-based methods could allow clinicians to establish more effective management and treatment of patients with suspected bloodstream infection at an earlier stage than is possible with only current culture-based approaches.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. "May 2007" Includes bibliographical references.

Axonal domains are required for proper neuron function. These domains are unstable and degenerate concurrent with the inflammation in multiple sclerosis (MS) and the inflammatory disease models experimental autoimmune encephalomyelitis (EAE) and lipopolysaccharide (LPS) induced inflammation. Previous studies from our laboratory have shown that the axon initial segment (AIS) is maintained independently of the presence of myelin, but that AIS disruption is seen in MS as well as EAE and LPS-mediated inflammation. AIS loss can be interrupted in the early stage of EAE using the anti-inflammatory drug Didox. However, the potential for Didox directed repair of the AIS in later stages of disease has not been investigated. Here, we utilize two models of CNS inflammation to assess the possibility of reversing AIS pathology. Based on our findings, we present the first evidence that AIS degeneration, an axonal pathology observed in MS and in chronic inflammation, is reversible.

Acute Lung injury (ALI) and the more severe acute respiratory distress syndrome (ARDS) are respiratory maladies that present immense clinical challenges. ALI affects 200,000 individuals annually and features a 40% mortality rate. ALI can be initiated by both pathogenic and sterile insults originating locally in the lungs or systemically. While immense research has been poured into this disease in an effort to find a therapeutic strategy, the heterogeneously diffuse nature of the disease has not yielded a cure for the disease. Death from this disease is strongly attributed to reduced gas exchange from a severely compromised alveolar-capillary barrier. The only way currently to manage this disease is through enhanced ventilation and hyperoxic therapy. Hyperoxic therapy is a common treatment given to over 800,000 patients each year to treat respiratory maladies such as ALI. Prolonged exposure to oxygen at high concentrations results in the development of a condition known as hyperoxic acute lung injury (HALI). In this disease, the formation of reactive oxygen species damages healthy tissue and impairs gas exchange. Hyperoxia is also a well-documented murine sterile lung injury model that replicates the symptoms of ALI in lung injury patients. The ability of non-lethal dosages of hyperoxia to resolve without lung fibrosis also enables the study of molecules associated with ALI resolution and repair, a process not clearly understood. Inflammation in ALI is associated with disease progression, however pharmaceutical interventions aimed at targeting the inflammatory cascade have failed in clinical trials for ALI. Recent reports point to an aberrant injury resolution mechanisms that may be more strongly correlated with morbidity and mortality. There seems to be a homeostatic imbalance between endogenous inflammation progression and resolution initiation. This is especially the case with HALI, as significant ROS generation results in depletion of redox regulating antioxidants. Resolution mechanisms associated with ALI in the oxygen toxicity setting is poorly understood. Polyunsaturated fatty acids such eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are essential fatty acids that show immense antioxidant and anti-inflammatory action in cases of acute injury. The lung mucosa is rich in DHA and following inflammatory insult DHA is readily converted to resolution phase interaction products (resolvins), which have shown immense proresolutionary potential in recent reports of acute injury. In the presence of aspirin, more potent and longer-acting aspirin-triggered resolvins are formed. The effects of resolvins and their aspirin triggered epimers have not been studied in an oxygen toxicity setting and are the focus of this dissertation. For the first time, we show that one of these resolvin molecules, aspirin triggered resolvin d1 (AT-RvD1), can enhance resolution of hyperoxic acute lung injury. In vitro results reveals that AT-RvD1 treatment resulted in reduced interaction of two key players in the HALI inflammatory cascade, the macrophage and alveolar epithelium. AT-RvD1 was able to blunt macrophage cytokine secretion as well as inhibit epithelial cell cytokine secretion and adhesion molecule expression. More importantly, AT-RvD1 blunted cytokine mediated leukocyte-epithelial cell interaction in vitro. In a sublethal hyperoxic injury model, mice given AT-RvD1 following hyperoxia exposure displayed reduced HALI pathological severity. ATRvD1 treatment resulted in reduced alveolar-capillary permeability, tissue inflammation, proinflammatory mediator secretion, epithelial cell death, and leukocyte influx. Taken together these novel results demonstrate the therapeutic potential of resolvins in the oxygen toxicity setting. These results also arouse the idea that resolvins could be used to lessen the comorbidities associated with oxygen therapy and improve recovery times of ALI patients.

Introdução: A Bronquiolite Obliterante Pós-infecciosa (BOPI) é uma síndrome clínica rara e grave caracterizada por sinais e sintomas persistentes de obstrução crônica das pequenas vias aéreas. Objetivos: o objetivo do presente estudo é avaliar a evolução da função pulmonar de uma coorte de acompanhamento de pacientes com BOPI de Porto Alegre, Brasil. Métodos: Foram incluídas crianças, adolescentes com BOPI, de ambos os sexos, em acompanhamento de longo prazo nos ambulatórios de pneumologia pediátrica dos hospitais da Criança Santo Antônio e Materno-Infantil Presidente Vargas, ambos de Porto Alegre, Brasil. Resultados: Quanto à análise das variáveis da função pulmonar, as médias globais dos pontos analisados no tempo para cada variável foram: da CVF foi de 68,8% ± 17,7, do VEF1 foi 48% ± 15, do VEF1/CVF foi 66% ± 17, e do FEF25-75 foi 25,4% ± 14. Longitudinalmente, encontramos que houve melhora estatística e clínica, significativa, da CVF (p=0,04). Já as variáveis VEF1 e o FEF25-75, que refletem melhor o componente obstrutivo, não apresentaram mudanças significativas tanto estatisticamente (p=0,708 e p=0,873 respectivamente) quanto clinicamente, mantendo valores similares no percentual do predito ao longo do tempo. A relação VEF1/CVF sofreu uma mudança estatisticamente significativa (p=0,015), clinicamente explicável pela maior melhora da CVF comparado com o VEF1. Conclusões: Em conclusão, os resultados do nosso estudo sugerem que, em crianças e adolescentes com bronquiolite obliterante pós-infecciosa, o componente obstrutivo da função pulmonar (VEF1 e FEF25-75) sofre poucas modificações significativas, deletérias ou benéficas, ao longo do tempo. A capacidade vital forçada, por outro lado, sofre um aumento progressivo ao longo do tempo que pode ser de grande importância como fator de proteção quando iniciar o declínio fisiológico da função pulmonar na idade adulta.
Introduction: The Post-Infectious Bronchiolitis Obliterans (PIBO) is a rare and serious clinical syndrome characterized by persistent signs and symptoms of small airways’ chronic obstruction. Objectives: The objetive of this study is evaliate the evoluction of pulmonary function in a monitoring cohort of patients with PIBO from Porto Alegre, Brazil. Methods: Children, adolescents with PIBO, of both sexes were included, in long-term monitoring in pediatric pneumology clinics of Santo Antônio children’s hospital and Presidente Vargas maternal and child hospital, both from Porto Alegre, Brazil. Results: Regarding the analysis of the variables of pulmonar function, the global average of ten points analyzed in the time for each variable were: from CVF was 68,8% ± 17,7, from VEF1 was 48% ± 15, from VEF1/CVF was 66% ± 17 and from FEF25-75 was 25,4% ± 14. Lengthwise, we found that there was an statistical and clinical improvement, significant, from CVF (p=0,04). However, the variables VEF1 and the FEF25-75, which better reflect the osbstructive component, don’t show significant changes as statistically (p=0,708 and p=0,873 respectively) as clinically, keeping similar values in percentage of the predicted over time. The relation VEF1/CVF suffered a statistically significant change (p=0,015), clinically explained by the greatest improvement in CVF compared with VEF1. Conclusões: In conclusion, the results of our study suggest that, in children and adolescents with post-infectious bronchiolitis obliterans, the obstructive component of pulmonary function (FEV 1 and FEF 25-75) suffers few significant changes, deleterious or beneficial, over time. The forced vital capacity, on the other hand, suffers a progressive increase over time which can be of great importance as protective factor when start the physiological decline of pulmonary function in adult age.

Lors de l'assemblage et du bourgeonnement du VIH, la membrane plasmique subit une courbure entraînée par l'auto-assemblage du VIH-1 Gag au site d'assemblage vers l'extérieur de la cellule. Cependant, la multimérisation de Gag peut ne pas être suffisante et Gag peut avoir besoin de recruter des facteurs cellulaires pour induire une courbure de membrane locale. Nous avons récemment rapporté que la libération de particules de VIH-1 Gag dépendait de l'activation de la voie de signalisation Rac1 / IRSp53 / Wave2 / Arp3 dans les cellules Jurkat T et les lymphocytes sanguins primaires. Ce complexe cellulaire, lorsqu'il est activé, est recruté dans la membrane plasmique cellulaire et favorise le recrutement de la ramification de l'actine, la polymérisation de l'actine et le remodelage de la membrane dans les lamellipodes. En particulier, la protéine IRSp53 contient un domaine I-BAR capable d'induire une courbure membranaire via la reconnaissance du phospholipide de membrane plasmique PI (4,5) P2. Ce phospholipide est également spécifiquement reconnu par le domaine Matrix N-terminal de Gag et est un cofacteur lipidique du ciblage de Gag vers la membrane plasmique et de l'assemblage du VIH-1. Ce projet de recherche visait à caractériser les mécanismes cellulaires et moléculaires de l'implication de l'IRSp53 dans l'assemblage du VIH-1 dans les cellules T CD4 et HEK293T. Nos résultats montrent que l'IRSp53 est associé à des particules virales et que son renversement par l'ARNsi diminue la libération du VIH-1 dans les lymphocytes T et les cellules T HEK293. La microscopie électronique des cellules renversées pour IRSp53 a révélé un phénotype frappant de bourgeons viraux arrêtés à un stade précoce de l'assemblage. L'immunoprécipitation de l'IRSp53 a montré une conversion indépendante de p6 de HIV-1 Gag, indiquant une complexation intracellulaire de Gag et de l'IRSp53. Le fractionnement cellulaire et la flottation de la membrane ont montré que le recrutement de l'IRSp53 vers la membrane cellulaire double lors de l'expression du VIH-1 Gag. La microscopie PALM / STORM à molécule unique bicolore et les analyses ultérieures ont montré l'IRSp53 à proximité des amas Gag. Nous avons également trouvé une incorporation spécifique de l'IRSp53 dans les particules de VIH-1 Gag par rapport à d'autres protéines I-BAR, un phénomène dépendant de son domaine I-BAR. Comme le domaine I-BAR est impliqué dans la courbure de la membrane, nous avons ensuite sondé cet aspect de l'implication de l'IRSp53 dans l'assemblage du VIH-1. L'analyse des images de microscopie électronique a révélé un défaut de courbure pour les bourgeons des cellules assommées IRSp53. Des études concomitantes dans des systèmes GUV in vitro sans cellule ont également indiqué un rôle pour la courbure de la membrane induite par IRSp53 dans la liaison à la membrane Gag. Ces résultats confirment le rôle essentiel de l'IRSp53 dans les premiers stades de l'assemblage du VIH-1. Enfin, comme l'IRSp53 est un acteur essentiel dans l'échafaudage des protéines de signalisation de l'actine, nous avons établi un rôle pour le Rac1 activé dans le recrutement de la membrane IRSp53 en aval du VIH-1 Gag, et testé l'implication du RacGEF Tiam1 dans la production de particules du VIH-1. La microscopie à super résolution révèle également la présence de nanostructures d'actine sur les sites d'assemblage du VIH-1. Tous ces résultats mettent en évidence le rôle nouveau et essentiel de la protéine I-BAR à courbure membranaire IRSp53, et la mobilisation de l'actine corticale, dans les phases tardives de la réplication du VIH-1
During the HIV assembly and budding, the plasma membrane undergoes a curvature driven by HIV-1 Gag self-assembly at the assembly site towards the exterior of the cell. However, the multimerization of Gag may not be sufficient and Gag may need to recruit cell factors for inducing local membrane curvature. We recently reported that the HIV-1 Gag particle release was dependent on the activation of the signalling pathway Rac1/IRSp53/Wave2/Arp3 in Jurkat T cells and primary blood lymphocytes. This cellular complex, when activated, is recruited to the cell plasma membrane and promotes the recruitment of actin branching, actin polymerisation and membrane remodelling in lamellipodia. In particular, the protein IRSp53 contains an I-BAR domain capable of inducing membrane curvature via the recognition of the plasma membrane phospholipide PI(4,5)P2. This phospholipid is also specifically recognized by the N-terminal Matrix domain of Gag and is a lipidic cofactor of Gag targeting to the plasma membrane and of HIV-1 assembly. This research project aimed at characterizing the cellular and molecular mechanisms of IRSp53 involvement in HIV-1 Gag assembly in CD4 T cells and HEK293T cells. Our results show that IRSp53 is associated with viral particles and that its knockdown by siRNA decreases HIV-1 release in T lymphocytes and HEK293 T cells. Electron microscopy of cells knocked down for IRSp53 revealed a striking phenotype of viral buds arrested at an early stage of assembly. Immunoprecipitation of IRSp53 showed a p6 independent pulldown of HIV-1 Gag, indicating intracellular complexing of Gag and IRSp53. Cellular fractionation and membrane flotation showed that IRSp53 recruitment to cellular membrane doubles upon expression of HIV-1 Gag. Dual colour single molecule PALM/STORM microscopy and subsequent analyses showed IRSp53 in close proximity to Gag clusters. We also found specific incorporation of IRSp53 in HIV-1 Gag particles as compare to other I-BAR proteins, a phenomenon dependent on its IBAR domain. As the I-BAR domain is involved in membrane curvature, we then probed this aspect of IRSp53 involvement in HIV-1 assembly. Analysis of electron microscopy images revealed a curvature defect for buds from IRSp53 knocked out cells. Concomitant studies in cell free in vitro GUV systems also indicated a role for IRSp53 induced membrane curvature in Gag membrane binding. These results affirm the essential role of IRSp53 in the early stages of HIV-1 assembly. Finally as IRSp53 is a vital player in scaffolding actin signaling proteins, we established a role for activated Rac1 in IRSp53 membrane recruitment downstream of HIV-1 Gag, and test the involvement of the RacGEF Tiam1 in HIV-1 particle production. Super resolution microscopy also reveals the presence of actin nanostructures at HIV-1 assembly sites. All these results highlight the novel and essential role of the membrane curving I-BAR protein IRSp53, and the mobilization of cortical actin, in the late phases of HIV-1 replication

Les ribosomes sont des complexes ribo-nucléoprotéiques qui lisent l'ARNm pour synthétiser les protéines. Il a été démontré que les inhibiteurs de la synthèse des protéines eucaryotes ont un potentiel thérapeutique important pour traiter un large éventail de cancers humains. Récemment, nous avons publié une étude multidisciplinaire dans laquelle nous démêlons le mécanisme d'action du chlorolissoclimide (CL), un composé qui partage la similarité chimique avec le cycloheximide (CHX), mais qui présente une cytotoxicité inférieure prometteuse. Nous nous sommes intéressés aux nouveaux types d'interactions de CL avec le ribosome et nous avons donc résolu une nouvelle structure cristalline du complexe S.cerevisiae 80S en complexe avec un autre composé de lissoclimide à une résolution de 3.1 Å. Le couplage de structures à haute résolution avec la conception de médicaments axée sur le calcul et l'analyse FRET guidera davantage la conception d'inhibiteurs plus sélectifs et moins cytotoxiques
Ribosomes are ribo-nucleoprotein complexes that read mRNA to synthesize proteins. This is important to consider in the case of cancer cells, which show a global increase in protein synthesis to support their hyper-proliferative behaviour. Inhibitors of eukaryotic protein synthesis have been shown to have significant therapeutic potential to treat a wide range of human cancers. Recently we published a multidisciplinary study in which we unravel the mechanism of action of chlorolissoclimide (CL), a compound sharing chemical similarity to cycloheximide (CHX), but showing promising lower cytotoxicity. We were interested in the new types of interaction of CL with the ribosome and therefore we solved a new crystal structure of the S.cerevisiae 80S in complex with another lissoclimide compound at 3.1 Å resolution. The coupling of high-resolution structures with computationally-driven drug design and FRET analysis will further guide the design of more selective and less cytotoxic inhibitors

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.

Au cours de ce travail de thèse expérimental, nous nous sommes intéressés à l’étude de la localisation nucléaire et nucléolaire de la protéine de la nucléocapside (NC) du VIH-1. Des études antérieures menées au laboratoire avaient mis en évidence une très forte accumulation de la NC dans les nucléoles. Ce compartiment nucléaire est connu pour être ciblé par de nombreux virus afin de promouvoir leur réplication. Des expériences de microscopie électronique avaient révélé la structure complexe du nucléole et montré qu’il est composé de trois sous-compartiments : les centres fibrillaires, le compartiment fibrillaire dense et le compartiment granulaire dans lesquels se déroule la synthèse des ribosomes. Afin de caractériser la localisation de la NC dans ces trois sous-compartiments, nous avons développé une approche de microscopie optique à haute résolution permettant d’obtenir des images à deux couleurs avec une résolution spatiale améliorée. Pour cela, nous avons mis au point un protocole qui permet d’utiliser simultanément une protéine fluorescente photocommutable et un fluorophore organique introduit par immunomarquage. Après avoir minimisé les aberrations optiques et corrigé les dérives mécaniques inhérentes au montage, nous avons visualisé simultanément la localisation de la NC surexprimée dans des cellules HeLa avec des marqueurs spécifiques des trois sous-compartiments nucléolaires (immunomarquage). La microscopie de fluorescence à haute résolution a permis de résoudre pour la première fois les différents compartiments et de montrer que la NC se localise préférentiellement dans le compartiment granulaire. Finalement, des expériences préliminaires avec des cellules vivantes ont permis de mettre en évidence que la NC est transportée de manière active dans le noyau et qu’elle pourrait interagir directement avec des protéines nucléolaires
During this experimental thesis work, we investigated the nuclear and nucleolar localization of the nucleocapsid protein (NC) of HIV-1. Previous studies performed in our laboratory evidenced a strong accumulation of NC in a subnuclear structure called nucleolus. Playing role in multiple cellular processes, nucleolus is often targeted by viruses to promote their replication. Electron microscopy revealed three nucleolar components (fibrillar centers, dense fibrillar component and granular component) associated to specific steps of the ribosome biogenesis. To characterize the distribution of the NC in these three sub-compartments and therefore shed light on the nucleolar localization of NC during the replication cycle, we developed a high-resolution optical microscopy approach. After having minimized the optical aberrations and corrected the mechanical drifts inherent to the imaging setup, the NC-mEos2 fusion protein overexpressed in HeLa cells was visualized simultaneously with immunolabeled nucleolar markers. The use of high-resolution fluorescence microscopy enabled us to resolve for the first time the three nucleolar compartments and to demonstrate the preferential localization of NC in the granular compartment of nucleolus. Finally, preliminary experiments performed with living cells showed that NC is actively transported in the nucleus and therefore may interact directly with nucleolar proteins

The human hepatitis B virus (HBV) is the prototype member of the Hepadnaviridae family of viruses. Various other hepadnaviruses are used as models to study human HBV infections as all Hepadnaviridae family members have similar virus structure and replication strategies. The studies performed and described in this thesis were carried out using duck hepatitis B virus (DHBV) infection of Pekin ducks as a model system. Hepadnavirus infections can have either an acute or a chronic outcome. The factors that contribute to these outcomes include the immune response, the age of the host at the time of infection as well as size of viral inoculum. The overall aim of this project was to gain a detailed understanding of the mechanisms involved in clearance of virus and resolution of acute DHBV infections. As a first step, molecular and immunohistochemical detection methods for a range of cellular markers in ducks had to be developed as assays were not readily available. Quantitative reverse transcription PCR assays (qRT-PCR) were developed for the detection of mRNA of the duck T-lymphocyte markers, CD3, CD4, CD8, duck cytokines, IFN-α, IFN-γ, TNF-α and the duck housekeeping genes, β-actin and GAPDH. Immunohistochemistry was developed for the detection of duck CD4 + and CD8 + on T cells and for the detection of proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation. These methods were then widely used throughout the project. The innate immune response during HBV infections is not well understood. Toll-like receptors (TLR) are a family of pattern recognition receptors that form part of the innate immune response and are involved in the recognition of bacterial, fungal and viral pathogens. The only TLR that have been reported to recognise viral pathogens are TLR- 2, TLR-3, TLR-4, TLR-7 and TLR-9. The possible role of TLR during hepadnavirus infections had not been well characterized to date. In this project cDNA sequences for duck TLR-2, TLR-4 and TLR-7 were identified and characterised and qRT-PCR assays were developed for their detection. Changes in duck TLR-2, TLR-4 and TLR-7 mRNA expression during hepadnavirus infection were identified following DHBV infection of primary duck hepatocytes (PDH) in vitro. The results showed increased levels of expression of duck TLR early during infection indicating an involvement of TLR and the innate immune response during DHBV infection. During the in vivo DHBV infection studies performed to date TLR mRNA expression remained unchanged. As previously mentioned hepadnavirus infection can have an acute or chronic outcome. We aimed to understand the mechanisms involved during the resolution of acute DHBV infection and to elucidate specific factors contributing to the successful resolution of infection. During acute infections immune markers were monitored by qRT-PCR and histological analysis of fixed liver sections was performed. Liver sections were analysed to detect liver inflammation, the number and size of Kupffer cells, hepatocyte apoptosis and changes in hepatocyte proliferation throughout the course of acute DHBV infection in 6-week-old ducks. By determining the percentage of DHBV-positive hepatocytes two patterns of clearance of acute DHBV infection were observed; early clearance of infected hepatocytes occurring before day 14 post infection (p.i.), and late clearance occurring after day 14, but before or on day 31 p.i. This viral clearance was seen to occur in a cell by cell pattern. Higher levels of hepatocyte proliferation and apoptotic hepatocytes were detected during the clearance phase (on day 14 p.i.) of the late clearance group. Periodic acid schiff-diastase (PAS-D) staining was used to show significant increases in both cell number and size of Kupffer cells. Levels of IFN-γ mRNA increased significantly over the uninfected age-matched control ducks on day 14 p.i. Levels of CD3, CD4 and CD8 mRNA expression also increased over the uninfected controls on days 14 and 31 p.i. In summary, we found that resolution of acute DHBV infection occurred on a cell by cell pattern of clearance, it was accompanied by increases in hepatocyte proliferation, apoptotic hepatocytes and activated Kupffer cells, indicating that T lymphocytes and cell death play important roles in the rapid clearance of DHBV infection. Following resolution of acute hepadnaviral infections residual viral DNA has been found to persist. Residual HBV DNA in humans can result in reactivation of HBV infection following liver transplantation or immunosuppressive drug treatment. This leads to possible pathogenic outcomes thus the need for further investigations. Previous studies performed in the duck model have shown that the major form of residual DNA is present as covalently closed circular DNA (cccDNA). We aimed to understand how this residual cccDNA was being maintained and if replication was involved in the process. Following resolution of infection in ducks, levels of residual DHBV DNA were monitored by quantitative PCR (qPCR). Ducks were treated with the Bristol-Myers Squibb nucleoside analogue Entecavir (ETV) in order to suppress any possible replication that might be maintaining levels of residual cccDNA. In DHBV-infected but non-ETV treated ducks, the levels of residual DHBV DNA decreased gradually when measured on days 60, 221 and 316 p.i. The observed decrease in residual DHBV DNA occurred in parallel with decreases in the rate of hepatocyte proliferation measured by PCNA staining. This finding suggests that levels of residual DHBV DNA and hepatocyte proliferation are linked and we hypothesise that hepatocyte turnover is involved in the clearance of residual DHBV DNA. ETV treatment did not have an effect on the levels of residual DHBV DNA which suggests that it is present in a subset of long-lived hepatocytes that do not support virus replication. Mathematical modelling was performed to predict the rate of hepatocyte proliferation required for the elimination of residual cccDNA. The mathematical modelling showed that the predicted rate of hepatocyte proliferation was consistent with the rate of hepatocyte proliferation measured by PCNA. Further mathematical modelling showed that residual cccDNA is most likely to survive mitosis and it decreases due to several rounds of hepatocyte proliferation required for its elimination. Altogether, this project has elucidated mechanisms involved during the resolution of acute DHBV infection and also possible mechanisms by which residual DHBV DNA is maintained following resolution of infection. Detailed understanding of the virological and immunological events that occur during the resolution of an acute hepadnavirus infection would assist in the development of new therapeutic treatments for the cure of chronic HBV infections.
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Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2008

博士
長庚大學
臨床醫學研究所
103
Sepsis remains to be the leading cause of mortality in critical care patients. Early identification of causative pathogen in sepsis patients can improve clinical outcome, and the current gold standard is to use blood culture. However, blood culture is often not effective in identifying the pathogens in three common types of sepsis patients: (I) patients that have been recently treated with antibiotics before blood culture; (II) patients that have cytokine disorder instead of microbial infection; (III) patients that are infected with pathogens that are not easily cultured. Even if blood culture can identify the causative pathogen, it is rather time consuming, and often requires 48-72 hours to identify the microbial. Due to the above problems, clinicians often rely on empirical antibiotic treatment modalities for sepsis patients. This is because the risk of mortality increases substantially in hourly increment when the appropriate antimicrobial therapy is delayed. Although use of empirical antibiotic can be effective, it can instead cause an emergence of drug-resistant organisms. Thus, my goal is to establish a rapid effective diagnostic tool for bloodstream infections, and thereby help clinicians select the most appropriate antibiotic treatment for sepsis patients. My research consists of two different parts. The first part is the establishment of a new innovative molecular diagnostic technique for microbial identification. To quantitatively identify microbial, I have combined real-time polymerase chain reaction (PCR) and high-resolution melting (HRM) technology. Using slightly different approaches, I have successfully identified 25 clinical common pathogens using this platform: 9 bacterial species can be identified via a 1-step post-PCR high-resolution melting analysis; 12 bacterial species can be identified via the high-resolution melting plots obtained by heteroduplex formation between the PCR products of the tested and reference bacterial species; and 4 bacterial species can be identified by a 2nd real-time PCR targeting a different region of the 16S ribosomal ribonucleic acid (rRNA) gene. The second part of my thesis is to solve bacterial deoxyribonucleic acid (DNA) contamination in PCR reagents. To solve contamination, we have employed broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. Broad-range PE-PCR amplification of the 16S rRNA gene can be validated and minute quantities of template DNA (10 femtogram) was detectable without false positives.

Multiorgan dysfunction syndrom is the major driving pathophysiological mechanism of morbidity and mortality in critically ill patients. Gastrointestinal dysfunction usually develops as a result critical illness and it is believed to play a key role in the development and progression of multiple organ dysfunction. Moreover, any primary disorder of the gastrointestinal tract, if severe enough, can lead to a critical state and secondary multiorgan dyfunction. Despite intensive experimental and clinical research, reliable tools for monitoring and evaluation of the severity of gastrointestinal dysfunction remain unknown. In the same line, therapy of this complex pathology remains largely supportive. The aim of this thesis was first to explain the severity of the most common and most serious nosocomial infection of the digestive tract, second to elucidate the safety and effectiveness of the endoscopic dual enteral probe insertion in ventilated critically ill patients, and, third to evaluate new diagnostic tools of the gastrointestinal dysfunction. Finally, we present an ongoing project aimed at investigating esophageal dysfunction in mechanically ventilated critically ill patients.

La majorité des individus exposés au virus de l’hépatite C (VHC) développent une infection chronique. Une réponse immunitaire adaptative forte et soutenue est associée avec la guérison spontanée du VHC, mais les mécanismes sous-jacents demeurent mal définis. Le rôle des cellules NK et des cellules dendritiques (DC) dans la guérison spontanée du VHC est encore méconnu. Les cellules NK sont la population effectrice la plus importante de l’immunité innée car elles tuent les cellules infectées et sécrètent diverses cytokines. Les DC reconnaissent des agents infectieux et elles sont les premières à initier et réguler l’immunité adaptative. Les cellules NK et les DC interagissent également entre elles afin de réguler l’immunité innée et adaptative. L’hypothèse du projet de doctorat est que l'activité des cellules NK pendant la phase aiguë de l'infection par le VHC module la fonction des DC afin que ces dernières puissent générer une réponse immunitaire adaptative capable d'éliminer le VHC. Le premier objectif était d’établir une corrélation entre l'activité des cellules NK et l'évolution de l'infection au VHC. Nous avons observé une augmentation de la cytotoxicité, mais une diminution de la sécrétion de cytokines par les cellules NK chez les patients chroniques et qui ont résolu spontanément pendant la phase aiguë en comparaison aux contrôles non infectés, démontrant alors une dissociation entre ces deux fonctions. Nos résultats suggèrent que les cellules NK sont activées pendant la phase aiguë indépendamment de l’évolution de l’infection. Le deuxième objectif était d’établir une corrélation entre le phénotype et la fonction des DC, et l'évolution de l'infection. Nous avons d’abord observé que les DC plasmacytoïdes de tous les patients infectés ont un phénotype plus immature que les contrôles, et que ce phénotype est plus prononcé chez les patients ayant résolu spontanément. De plus, en réponse à des stimulations, nous avons observé que pendant la phase aiguë précoce, les DC myéloïdes (mDC) de tous les patients infectés indépendamment de l’évolution de l’infection produisent davantage de cytokines en comparaison aux contrôles. Cependant, cette hyperréactivité n’est pas soutenue au cours de l’évolution chronique. Le troisième objectif était d’établir une corrélation entre les interactions NK/DC et l’évolution de l’infection. Nous avons étudié la capacité des cellules NK à lyser les DC potentiellement tolérogéniques, ainsi que la capacité des DC matures à activer les cellules NK, et nous avons observé aucune différence entre les patients infectés et les contrôles. Finalement, nous avons démontré pour la première fois la capacité des DC immatures à inhiber la fonction des cellules NK. En conclusion, nous avons démontré que les cellules NK sont activées pendant la phase aiguë de l’infection par le VHC indépendamment de l’évolution de l’infection. De plus, la capacité des cellules NK à éliminer les DC potentiellement tolérogéniques est intacte. Finalement, les mDC sont hyperréactives pendant la phase aiguë de l’infection, mais cette hyperréactivité n’est pas soutenue avec la persistance de l’infection. Cette perte d’hyperréactivité des mDC ne semble pas affecter la capacité des DC à activer les cellules NK, mais elle pourrait jouer un rôle dans l’inefficacité de l’immunité adaptative à éliminer le VHC.
The majority of individuals exposed to the hepatitis C virus (HCV) develop a chronic infection. It is known that a strong and sustained adaptive immune response is associated with the spontaneous clearance of HCV, however the underlying mechanisms are not well defined. The role of natural killer (NK) cells and dendritic cells (DCs) during the spontaneous resolution of HCV remains unknown. NK cells are the primary effector population of the innate immune response which are able to kill infected cells and secrete various cytokines. On the other hand, DCs are the first cell type to initiate and regulate adaptive immunity after recognizing infectious pathogens. NK cells and DCs can also interact reciprocally to further regulate innate and adaptive immunity. Our hypothesis is that NK cell activity during acute HCV will modulate DC function to prime a highly efficient adaptive immune response resulting in viral clearance. The first aim of my project was to establish a correlation between NK cell activity and the outcome of HCV infection. We observed increased NK cell cytotoxicity, but decreased cytokine secretion during acute HCV in patients with chronic evolution as well as spontaneous resolution, further demonstrating a dissociation between these two NK cell functions. Our results suggest that NK cells are activated during acute HCV infection regardless of infection outcome. The second aim was to establish a correlation between DC phenotype, function and the outcome of infection. We observed that plasmacytoid DCs (pDCs) from all HCV-infected patients have a more immature phenotype as compared to negative controls, yet this is more pronounced in spontaneous resolvers. Furthermore, we observed that during the early acute phase, myeloid DCs (mDCs) from all HCV-infected patients, regardless of outcome, have increased production of cytokines as compared to un-infected controls in response to stimulation. However, this hyperresponsiveness of mDCs is not sustained with chronic evolution. The third aim was to establish a correlation between the NK/DC cross-talk and infection outcome. We studied the capacity of NK cells to kill potentially tolerogenic DCs, as well as the capacity of mature DCs to activate NK cells, and we observed no major differences between different stages of HCV infection and un-infected controls. However, we obtained unprecedented data which suggests that immature DCs have the capacity to inhibit NK cell function. In conclusion, our results demonstrate that NK cells are activated during acute HCV infection regardless of its outcome. Furthermore, the capacity of NK cells to kill potentially tolerogenic DCs is intact for all groups of patients. Finally, mDCs are hyperresponsive during acute HCV, but this hyperresponsiveness is not sustained with persistence of viremia. The loss of mDC hyperresponsiveness does not seem to affect the capacity of DCs to activate NK cells, but might play a role in the capacity of DCs to prime a highly efficient adaptive immune response resulting in viral clearance.

This qualitative research sought to examine the actual impact of HIV/AIDS on the human security of households in Bulawayo. The two research questions in this study where, how does HIV/AIDS affect the seven areas of human security? and has the economic crisis in Zimbabwe increased the impact of HIV/AIDS on households? This study utilized both primary and secondary data in which the later was consulted in constructing the literature review and to address specific aims one and two. These specific aims were: to assess the human security conditions in Zimbabwe and to examine the potential impact of HIV/AIDS on human security. Data was gathered in the form of two focus group discussions held in Bulawayo with faith-based support groups and also in the form of in-depth interviews with households which were not connected to the faith based support groups in Bulawayo. A total of 29 participants took part in this research representing 29 households. 19 of these households were represented in the two focus group discussions while the remainder of, 10 households were represented in the in-depth interviews. Human security is presented as different from traditional security in that the later seeks to protect nations from external threats while the former seeks to protect people from both external and internal threats such as threats of chronic diseases, hunger, unemployment, crimes, social conflicts, political repressions, environmental hazards and HIV/AIDS. These threats can be natural, manmade or both. Human security was assessed in light of the seven areas of threats to human security which are economic, food, health, environment, personal, community, and political security. Four major themes emanated from this research these being: the financial, health, nutritional and societal impacts of HIV/AIDS on the households in Bulawayo, chief of these being the financial impact in form of increased expenditure, reduced income and diverted investments of households. This study came up with recommendations that aim at reducing and ultimately eradicating the impact of HIV/AIDS on households these being economic empowerment, food aid, ARVs provisions, training in survival skills and orphan care. The major challenge was given to individuals, families, society and NGOs especially the FBOs to take the lead in implementing these recommendations as the government is not yet in a position to do so.
Thesis (M.Com.)-University of KwaZulu-Natal, Westville, 2009.

博士
國立陽明大學
生化暨分子生物研究所
96
Crystal Structure of Infectious Bursal Disease Virus VP2 Subviral Particle at 2.6 Å Resolution: Implications in Virion Assembly and Immunogenicity Abstract Infectious bursal disease virus (IBDV) is responsible for the hightly contagious and immunosuppressive disease in young chicken. The structural protein VP2 of IBDV spontaneously forms a dodecahedral T=1 subviral particle (SVP), and is a primary immunogen of the virus, understand of its structure is efficient for vaccine development. In this study, the structure of IBDV SVP was determined in a cubic crystal and refined to 2.6Å resolution. It contains 20 independent VP2 subunits in a crystallographic asymmetric unit. Each subunit is folded mainly into a shell domain and a protrusion domain, both with the Swiss-roll topology, plus a small helical base domain. Three VP2 subunits constitute a tight trimer, which is the building block of IBDV (sub)viral particles. The structure revealed a calcium ion bound to three pairs of symmetry-related Asp31 and Asp174 to stabilize the VP2 trimer. To investigate the effect of Ca2+ on the IBDV SVP structure, we used EGTA to remove the divalent ion and analyzed the particle morphology by gel electrophoresis and electron microscopy, and the results indicated that the metal-ion may be important not only in maintaining highly stable quaternary structure but also in regulating the swelling and dissociation of the icosahedral particles. A Ca2+-dependent assembly pathway was thus proposed, which involves further interactions between the trimers. The 20 independent subunits showed conformational variations, with the surface loops of the protrusion domain being the most diverse. These loops are targets of the neutralizing antibodies. Several common interactions between the surface loops were clearly observed, suggesting a possible major conformation of the immunogenic epitopes. Knowledge of the three-dimensional structure of SVP may be useful in rationally incorporating important foreign epitopes into the loop region to create engineered recombinant SVP as new potent immunogens or vaccines. Structural Basis of Metal-conjugated Complexes as 3C and 3C-like Protease Inhibitors Abstract Viral proteases have been pursued for anti-virus therapy, and their crystal structures were used to assist the design of inhibitors. Some metals (Cu2+, Hg2+, Zn2+) and metal-conjugated compounds showed cysteine protease inhibition activity. Here, to elucidate the metal-inhibitor binding mode and to synthesize better inhibitors, 3C-like protease from Coronaviridae and 3C protease from Piconaviridae complexed with metal-conjugated inhibitors were analyzed crystallographically. Five active metal-conjugated inhibitors (PMA, TDT, EPDTC, JMF1586 and JMF1600) bound with the 3C-like protease (3CLpro) of severe acute respiratory syndrome (SARS)-associated coronavirus (CoV) were determined. The complex structures reveal two major inhibition modes: Hg2+-PMA is coordinated to C44, M49 and Y54 with a square planar geometry in the S3 pocket, whereas each Zn2+ of the four zinc-inhibitors is tetrahedrally coordinated to the His-Cys catalytic dyad. 3CLpro of human coronavirus 229E (HCoV-229E) and 3C proteases of Coxsackie B viruses type 3 (CVB3) also have the His-Cys catalytic residues as 3CLpro of SARS-CoV. The first crystal structures of CVB3 3Cpro, and the crystal structure of 3Cpro from CVB3 and 3CLpro from HCoV-229E in complex with the inhibitor EPDTC were also determined. The zinc ion of EPDTC is again tetrahedrally coordinated to the His-Cys catalytic residues of CVB3 3Cpro and HCoV-229E 3CLpro. For anti-virus drug design, this Zn2+-centered coordination pattern would serve as a starting platform for inhibitor optimization.

Many people do not like thinking about the future. If they do, over 50% of Canadians think “our way of life” (p. 7) will end within 100 years and over 80% of Canadians think “we need to change our worldview and way of life if we are to create a better future for the world” (Randle & Eckersley, 2015, p. 9). There is a good reason for this. Alarms have sounded over global urgent complex problems with potential for catastrophic consequences such as the development of artificial intelligence, climate change, mass extinction, nuclear war and pandemics (Marien & Halal, 2011). Society is also increasingly fragmenting as imminent crises build on lack of understanding, the sense of incapacity to act, fear, distrust, blame and a lack of hope. This struggle for humanity’s survival is complicated by the turbulent global environment in which institutions continue to follow path-dependent trajectories set forth in a different time and context. Governments at various levels face a problem of “fit” between current structures and processes, that have not progressed sufficiently to meet changing needs of a global society mired in complexity and governance challenges. However, hope exists. Incremental progress on many fronts and a massive amount of efforts and resources are being engaged worldwide. There are emerging fields, lenses and tools that can potentially alleviate complex problems and address this emergency. The purpose of this dissertation is to understand and assess dialogue-based foresight practices being applied towards complex problems in Canada to provide insights into how these practices can assist society to alleviate global urgent complex problems and their impacts, within this backdrop of looming crises. Foresight, alternatively known as future studies or scenario-building, is a forward-looking practice recognized and used globally with over 100 research organizations focused on foresight, widespread usage by firms and over 18 countries involved in foresight activities (Berze, 2014b). Overall literature findings suggest foresight is widely and at least incrementally effective with a number of impacts in various areas (Calof, Miller, & Jackson, 2012; March, Therond, & Leenhardt, 2012; Meissner, Gokhberg, & Sokolov, 2013) but the extent of this effectiveness, the mechanisms involved, and the specific foresight benefits per type of project needs further research and evidence. For instance, limited literature exists on whether foresight can transform complex situations and if so, under what conditions. Thus, opportunities exist for assessing and increasing foresight’s impact. This dissertation is a contextualized, systematic empirical study that taps into transdisciplinary literature and practice, case studies of how foresight has been used to address specific types of complex problems in Canada, as well as surveys and interviews with foresight experts and participants. This dissertation uses a foresight community scan and a comparative case study approach to provide practical and theoretical benefits to foresight and complex problem area stakeholders. The research focuses on studying the broad interactions of foresight and identifying the impacts of dialogue-based foresight projects on people and the outcomes of complex problems. The dissertation concludes that dialogue-based foresight is a valuable and unique practice for ameliorating complex problems and their consequences. Insights are offered towards dialogue-based foresight’s potential contributions within the context of other efforts directed at humanity’s struggle for survival and global complex problems. These insights can then foster the further development and application of dialogue-based foresight on a global scale to alleviate complex problems and their effects. The dissertation outlines recommendations on key next steps to realize these potential contributions.
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