Lors des infections bactériennes, selon les tissus infectés, et selon la nature des pathogènes, l’organisme répond en mobilisant différents acteurs. Nous avons décidé d’utiliser le modèle du zebrafish ou Danio rério pour étudier la réponse immunitaire innée dans les situations d’infection bactérienne où les phagocytes professionnels ne peuvent pas venir au contact direct des bactéries. Pour cela, j’ai développé un modèle d’infection de la notochorde del’embryon de zebrafish. Lors de l’injection des bactéries dans ce compartiment, les bactéries se retrouvent protégées par une épaisse gaine de collagènes que les phagocytes ne peuvent pas pénétrer. Alors que les mycobactéries,protégées par la gaine de collagène ne sont pas détectées par les phagocytes, les bactéries E. coli sont immédiatement détectées ce qui déclenche une importante inflammation locale autour de la notochorde. Alors que les bactéries E. coli, bien qu’inaccessibles à la phagocytose sont éliminées dans les première 24 heures qui suivent l’injection, l’inflammation dure plusieurs jours.J’ai étudié les mécanismes qui conduisent à cette inflammation persistante et ses conséquences à long terme sur le développement du poisson. J’ai montré le rôle central de la cytokine IL1b dans ce processus, et j’ai développé une lignée transgénique qui permet d’étudier l’induction de cette cytokine in vivo chez le poisson.J’ai ensuite étudié le rôle des deux principales populations de phagocytes dans l’élimination des bactéries E coli. J’ai montré que les macrophages ne sont pas impliqués dans la disparition des bactéries alors que les neutrophiles, bien qu’incapable de pénétrer à l’intérieur de la gaine de collagène sont nécessaires à l’élimination des bactéries.J’ai ensuite montré que la myelopéroxidase et le monoxyde d’azote ne sont pas impliqués dans l’élimination des bactéries alors que les espèces réactives de l’oxygène produites par les neutrophiles sont nécessaires pour éradiquer l’infection. / In bacterial infections, according to the infected tissue and the nature of pathogens, the body responds by mobilizing various actors. I decided to use zebrafish or Danio rerio model to study the innate immune response to bacterial infection in the situations that professional phagocytes cannot come in direct contact with the bacteria. For this, I developed a model of infection in the notochord of zebrafish embryo. Upon injection of bacteria in this compartment, the microbes find themselves protected by the thick collagensheath where the phagocytes cannot penetrate. While mycobacteria are not detected by phagocytes; E. coli bacteria are sensed and a significant local inflammation around the notochord is mounted. The E. coli, although inaccessible to phagocytosis are eliminated within the first 24 hours after injection, the inflammation lasts several days.I studied the mechanisms that lead to this persistent inflammation and its long term consequences on the development of the fish. I showed the central role of the cytokine IL1B in this process, and I developed a transgenic line that allows studying in vivo the induction of this cytokine in fish.I then studied the roles of the two main populations of phagocytes in the elimination of E. coli. I revealed that macrophages are not involved in the removal of bacteria but neutrophils, although unable to penetrate inside the collagen casing, are necessary for the bacterial elimination. I also confirmed that myeloperoxidase and nitrogen monoxide are not involved in the removal of bacteria, rather the reactive oxygen species produced by neutrophils are needed to eradicate the infection.

A conspicuous feature of plants responding to pathogen invasion is the synthesis of nitric oxide (NO), a redox signal. NO regulates protein function by S-nitrosylation, the addition of an NO moiety to a cysteine thiol to form an S-nitrosothiol. A key theme of NO function is reprogramming plant immune-related gene expression. However, it is still not clear how the NO signal is translated into transcriptional changes. Here we explored the potential role of a sub-group of SNO Regulated Genes (SRGs) uncovered by global expression profiling. Firstly, transgenic plants containing the SRG1 or SRG3 promoter fused to glucuronidase gene GUS together with qRT-PCR assays confirmed that transcripts of SRGs could be induced by NO and pathogen challenge, suggesting that SRGs may be involved in NO signalling related to plant immunity. More importantly, transient and stable overexpression of SRG genes induced hypersensitive response (HR)-like cell death development, which is often associated with pathogen effector-triggered immunity. Furthermore, transgenic plants constitutively expressing SRG genes exhibited enhanced ROS accumulation, PR1 transcript accumulation, and increased resistance to Pseudomonas syringae (Pst) DC3000 compared with Col-0 wild type plants. In contrast, lines with T-DNA insertions into SRG genes exhibited susceptibility to Pst DC3000. These data suggested SRGs act as the positive regulators in plant immunity. In order to further explore how NO regulates these SRGs in plant immunity, we focused on SRG1 and found SRG1 could be S-nitrosylated in vitro and in vivo. Moreover, electrophoretic mobility shift assays showed SRG1 could bind to an AGT motif and the transcriptional activity was blunted in the presence of NO, suggesting that the DNA binding activity of SRG1 is redox-modulated. Further, a transient repression activity assay showed that SRG1 has repression activity and this activity was impaired in the gsnor1-3 mutant, which has a high S-nitrosothiols level. These data suggested NO could block SRG1 transcriptional activity in vitro and in vivo. Furthermore when the SRG1 overexpression line was crossed with gsnor1-3 the SRG1-mediated resistance related phenotypes were suppressed. These data demonstrated NO negatively regulates SRG1 transcriptional activity during plant immunity. SRG1 may therefore be an important regulator of NO signalling and subsequent regulate transcription during plant immunity. Additionally, NO may negatively feedback to inhibit transcriptional activity of SRG1 to control its repression activity, to enable the activation of plant immunity.

D.Litt. et Phil. (Psychology) / Please refer to dull text to view abstract

M. Tech. / The persistent presence of neutrophils is associated with a wide range of inflammatory diseases. Resolution of inflammation in these diseases is also associated with the ingestion of apoptotic neutrophils by macrophages. Inflammation and apoptosis of inflammatory cells are common known features observed in the lung following exposure to crystalline silica. What is not known is how well these apoptotic cells are cleared by macrophages in the presence of crystalline silica? To investigate the latter, we incubated the U937 macrophages and neutrophils with crystalline silica and found that it could increase their apoptosis and necrosis especially those of the U937 cells. We then examined the ability of crystalline silica to induce the production of cytokines (TNF-α, IFN-γ and IL-1β) as well as NO by these cells. We found that these particles could increase the production of TNF-α, IL-1β and NO but not IFN-γ in a time-and concentration-dependent manner. We also assessed the ability of crystalline silica to alter the levels of GSH in neutrophils and U937 macrophages. We found that it could drastically decrease the levels of this antioxidant in U937 macrophages with no additional effect in neutrophils as these latter cells would have low levels of GSH prior to their incubation with crystalline silica. Finally, we examined the effect of crystalline silica on the ability of U937 macrophages to phagocytose apoptotic neutrophils. We found that while untreated U937 macrophages were able to phagocytose apoptotic neutrophils, the presence of crystalline silica reduced this ability by 15%. Taken together, our results suggest that exposure to crystalline silica impairs the clearance of apoptotic neutrophils by decreasing their phagocytosis by macrophages and thus prevents the resolution of inflammation.

La complexité de la prise en charge des hépatites B chroniques est en partie liée à la persistance virale. La clairance du virus est compliquée du fait du mode de réplication du virus et de l'interaction de HBV avec les acteurs de la réponse immunitaire innée. On distingue plusieurs stades d'infection chronique à HBV, qui se caractérisent par un équilibre variable entre réplication virale et réponse immunitaire de l'hôte. La définition clinique de ces différentes phases est insuffisante pour discriminer le risque évolutif des patients. Des études de cohorte sont nécessaires pour mieux appréhender les relations entre réplication virale et immunité lors des infections chroniques à HBV. Le premier travail est une étude de cohorte rétrospective s'intéressant aux relations entre marqueurs virologiques sériques et intrahépatiques et expression intrahépatique des gènes de l'immunité innée. Les gènes de l'immunité innée étudiés sont moins exprimés au cours des hépatites B chroniques en comparaison à des contrôles sains, sans influence de la réplication virale. Le taux d'AgHBs sérique pourrait refléter l'importance de la réponse immunitaire innée intrahépatique, notamment la réponse IFN de type I pour les patients négatifs pour l'AgHBe. Le deuxième travail est une étude de cohorte prospective, qui ne retrouve pas de bénéfice à l'addition d'un traitement par PEG-IFN chez des patients co-infectés HBV/HIV et répondant au traitement par NUCS. Cette étude souligne de façon intéressante que le taux d'AgHBs à l'initiation du traitement pourrait être prédictif de la réponse au traitement, des taux plus faibles d'AgHBs étant favorables. L'ensemble de ces résultats démontre l'importance de la relation entre réponse immunitaire et réplication virale pour expliquer les différentes phases de l'hépatite B chronique. Ces résultats pourraient se révéler intéressants pour le développement de nouvelles prises en charge thérapeutiques de patients infectés chroniquement par HBV / Chronic HBV infections (CHB) are difficult to treat diseases because of viral persistence. It’s explained by the particular replication of Hepatitis B Virus (HBV) and its interplays with host immunity. CHB is characterized by different stages, which reflect a balance between viral replication and immune response. However, our knowledge regarding the natural history of CHB is insufficient to allow us to predict patients’ prognosis. Further clinical studies are needed to improve our understanding of interplays between HBV replication and host immunity. The first study is a retrospective one about interplays between serological and intrahepatic viral markers and intrahepatic innate immunity genes expression. Immunity genes seem to be down-regulated during CHB in comparison to healthy controls, without impact of the level of viral replication. HBsAg levels in blood may reflect the intrahepatic innate immune response and especially the type I IFN response for HBeAg negative patients. The second part is a prospective study which shows any relevance of adding PEG-IFN to HIV/HBV co-infected responders to NUCS therapy patients. The results highlight the potential interest of baseline HBsAg level to predict PEG-IFN response (low HBsAg levels being more favorable). Finally, these results highlight the role of interplays between HBV replication and innate immune response during the natural history of CHB. They may be interesting in the context of the development of new antiviral strategies

Magister Legum - LLM / The International Criminal Court (hereafter ‘ICC’ or ‘the Court’), in its fight against impunity is slated to put on trial, in conformity with Article 27 of the Rome Statute1, an incumbent Head of State and his Deputy for crimes under Article 7 of the Statute. The President and Deputy President of the Republic of Kenya are currently accused of crimes against humanity before the ICC, for acts of violence perpetrated in the wake of the December 2007 presidential and parliamentary elections. This research is a study of the ICC’s conceptual framework and positive implementation of its mandate with respect to prosecuting Kenya’s top leaders. By critically evaluating the cases against President Kenyatta and Deputy President Ruto, this research aims to assess the feasibility of such high-profile cases which have, in part, contributed to the hostility surrounding the Court in its fight against impunity. To this end, the factual and legal background, as well as the political context of the cases will be discussed.

Magister Legum - LLM / This study is about the individual liability of councillors in South Africa. It examines and analyses whether individual liability of councillors can serve as a bulwark against the abuse of decision-making power that is vested in the municipal council.

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The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate