The global regulator GacA of Pseudomonas fluorescens CHA0 is required for the suppression of root diseases in dicotyledons but not in Gramineae

Pseudomonas fluorescens strain CHA0 suppresses various plant diseases caused by soil-borne fungi. The pseudomonad produces the antimicrobial metabolites 2,4-diacetylphloroglucinol (Phl), pyoluteorin (Plt) and hydrogen cyanide, which are important for disease suppression, as well as the siderophores pyoverdine (Pvd), salicylic acid (Sal) and pyochelin (Pch). In the current work, a derivative of CHA0 with a mutation in the global regulator gene gacA (GacA−), which is unable to produce Phl, Plt and HCN, failed to protect the dicotyledonous plants cress and cucumber against damping-off caused by Pythium ultimum. In contrast, the GacA− mutant could still protect the Gramineae wheat and maize against damping-off mediated by the same strain of P. ultimum, and wheat against take-all caused by Gaeumannomyces graminis. However, the GacA− mutant overproduced Pch and Pvd. To gain more insight into disease protection afforded by the GacA− mutant, a GacA− Pvd− double mutant (strain CHA496) was constructed by gene replacement. Strain CHA496 overproduced Pch and Sal compared with CHA0 and protected wheat against P. ultimum and G. graminis, whereas cress and cucumber were not protected. Addition of FeCl3 repressed Pch and Sal production by strain CHA496 in vitro and impaired the protection of wheat in soil microcosms. In conclusion, a functional gacA gene was necessary for the protection of dicotyledons against root diseases, but not for that of Gramineae. Results indicated also that Pch and/or Sal were involved in the ability of the GacA− Pvd− mutant of CHA0 to suppress root diseases in Gramineae.

Arabidopsis lox3 lox4 double mutants are male sterile and defective in global proliferative arrest.

Fertility and flower development are both controlled in part by jasmonates, fatty acid-derived mediators produced via the activity of 13-lipoxygenases (13-LOXs). The Arabidopsis thaliana Columbia-0 reference genome is predicted to encode four of these enzymes and it is already known that one of these, LOX2, is dispensable for fertility. In this study, the roles of the other three 13-LOXs (LOX3, LOX4 and LOX6) were investigated in single and double mutants. Four independent lox3 lox4 double mutants assembled with different mutated lox3 and lox4 alleles had fully penetrant floral phenotypes, displaying abnormal anther maturation and defective dehiscence. The plants were no longer self-fertile and pollen was not viable. Fertility in the double mutant was restored genetically by complementation with either the LOX3 or the LOX4 cDNAs and biochemically with exogenous jasmonic acid. Furthermore, deficiency in LOX3 and LOX4 causes developmental dysfunctions, compared to wild type; lox3 lox4 double mutants are taller and develop more inflorescence shoots and flowers. Further analysis revealed that developmental arrest in the lox3 lox4 inflorescence occurs with the production of an abnormal carpelloid flower. This distinguishes lox3 lox4 mutants from the wild type where developmentally typical flower buds are the terminal inflorescence structures observed in both the laboratory and in nature. Our studies of lox3 lox4 as well as other jasmonic acid biosynthesis and perception mutants show that this plant hormone is not only required for male fertility but also involved in global proliferative arrest.

Small Cities, Neoliberal Governance and Sustainable Development in the Global South : A Conceptual Framework and Research Agenda

Development and environmental issues of small cities in developing countries have largely been overlooked although these settlements are of global demographic importance and often face a "triple challenge"; that is, they have limited financial and human resources to address growing environmental problems that are related to both development (e.g., pollution) and under-development (e.g., inadequate water supply). Neoliberal policy has arguably aggravated this challenge as public investments in infrastructure generally declined while the focus shifted to the metropolitan "economic growth machines". This paper develops a conceptual framework and agenda for the study of small cities in the global south, their environmental dynamics, governance and politics in the current neoliberal context. While small cities are governed in a neoliberal policy context, they are not central to neoliberalism, and their (environmental) governance therefore seems to differ from that of global cities. Furthermore, "actually existing" neoliberal governance of small cities is shaped by the interplay of regional and local politics and environmental situations. The approach of urban political ecology and the concept of rural-urban linkages are used to consider these socio-ecological processes. The conceptual framework and research agenda are illustrated in the case of India, where the agency of small cities in regard to environmental governance seems to remain limited despite formal political decentralization.

New Findings in a Global Approach to Dissect the Whole Phenotype of PLA2G6 Gene Mutations.

Mutations in PLA2G6 gene have variable phenotypic outcome including infantile neuroaxonal dystrophy, atypical neuroaxonal dystrophy, idiopathic neurodegeneration with brain iron accumulation and Karak syndrome. The cause of this phenotypic variation is so far unknown which impairs both genetic diagnosis and appropriate family counseling. We report detailed clinical, electrophysiological, neuroimaging, histologic, biochemical and genetic characterization of 11 patients, from 6 consanguineous families, who were followed for a period of up to 17 years. Cerebellar atrophy was constant and the earliest feature of the disease preceding brain iron accumulation, leading to the provisional diagnosis of a recessive progressive ataxia in these patients. Ultrastructural characterization of patients' muscle biopsies revealed focal accumulation of granular and membranous material possibly resulting from defective membrane homeostasis caused by disrupted PLA2G6 function. Enzyme studies in one of these muscle biopsies provided evidence for a relatively low mitochondrial content, which is compatible with the structural mitochondrial alterations seen by electron microscopy. Genetic characterization of 11 patients led to the identification of six underlying PLA2G6 gene mutations, five of which are novel. Importantly, by combining clinical and genetic data we have observed that while the phenotype of neurodegeneration associated with PLA2G6 mutations is variable in this cohort of patients belonging to the same ethnic background, it is partially influenced by the genotype, considering the age at onset and the functional disability criteria. Molecular testing for PLA2G6 mutations is, therefore, indicated in childhood-onset ataxia syndromes, if neuroimaging shows cerebellar atrophy with or without evidence of iron accumulation.

Therapeutic drug monitoring and metabolites profil analysis of antiviral drugs

The widespread use of combination antiretroviral therapy (ARVs) has considerably improved the prognosis of patients infected with HIV. Conversely, considerable advances have been recently realized for the therapy of hepatitis C infection with the recent advent of potent new anti-HCV drugs that allow an increasing rate HCV infection cure. Despite their overall efficacy, a significant number of patients do not achieve or maintain adequate clinical response, defined as an undetectable viral load for HIV, and a sustained virological response (or cure) in HCV infection. Treatment failure therefore still remains an important issue besides drugs toxicities and viral resistance which is not uncommon in a significant percentage of patients who do not reach adequate virological suppression. The reasons of variability in drug response are multifactorial and apart from viral genetics, other factors such as environmental factors, drug- drug interactions, and imperfect compliance may have profound impact on antiviral drugs' clinical response. The possibility of measuring plasma concentration of antiviral drugs enables to guide antiviral drug therapy and ensure optimal drug exposure. The overall objective of this research was to widen up the current knowledge on pharmacokinetic and pharmacogenetic factors that influence the clinical response and toxicity of current and newly approved antiretroviral and anti-HCV drugs. To that endeavour, analytical methods using liquid chromatography coupled with tandem mass spectrometry have been developed and validated for the precise and accurate measurement of new antiretroviral and anti-HCV drugs . These assays have been applied for the TDM of ARVs and anti-HCV in patients infected with either HIV or HCV respectively, and co-infected with HIV- HCV. A pharmacokinetic population model was developed to characterize inter and intra-patient variability of rilpivirine, the latest marketed Non Nucleoside Reverse transcriptase (NNRTI) Inhibitor of HIVand to identify genetic and non genetic covariates influencing rilpivirine exposure. None of the factors investigated so far showed however any influence of RPV clearance. Importantly, we have found that the standard daily dosage regimen (25 mg QD) proposed for rilpivirine results in concentrations below the proposed therapeutic target in about 40% of patients. In these conditions, virologie escape is a potential risk that remains to be further investigated, notably via the TDM approach that can be a useful tool to identify patients who are at risk for being exposed to less than optimal levels of rilpivirine in plasma. Besides the last generation NNRTI rilpivirine, we have studied efavirenz, the major NNRTI clinically used so far. Namely for efavirenz, we aimed at identifying a potential new marker of toxicity that may be incriminated for the neuropsychological sides effects and hence discontinuation of efavirenz therapy. To that endeavour, a comprehensive analysis of phase I and phase II metabolites profiles has been performed in plasma, CSF and in urine from patients under efavirenz therapy. We have found that phase II metabolites of EFV constitute the major species circulating in blood, sometimes exceeding the levels of the parent drug efavirenz. Moreover we have identified a new metabolite of efavirenz in humans, namely the 8-OH-EFV- sulfate which is present at high concentrations in all body compartments from patients under efavirenz therapy. These investigations may open the way to possible alternate phenotypic markers of efavirenz toxicity. Finally, the specific influence of P-glycoprotein on the cellular disposition of a series ARVs (NNRTIs and Pis] has been studies in in vitro cell systems using the siRNA silencing approach. -- Depuis l'introduction de la thérapie antirétrovirale (ARVs) la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Une réponse imparfaite ou la toxicité du traitement est certainement multifactorielle. Le suivi thérapeutique des médicaments [Therapeutic Drug Monitoring TDM) à travers la mesure des concentrations plasmatiques constitue une approche importante pour guider le traitement médicamenteux et de s'assurer que les patients sont exposés à des concentrations optimales des médicaments dans le sang, et puissent tirer tout le bénéfice potentiel du traitement. L'objectif global de cette thèse était d'étudier les facteurs pharmacocinétiques et pharmacogénétiques qui influencent l'exposition des médicaments antiviraux (ARVs et anti- VHC) récemment approuvés. A cet effet, des méthodes de quantification des concentrations plasmatiques des médicaments antirétroviraux, anti-VHC ainsi que pour certains métabolites ont été développées et validées en utilisant la Chromatographie liquide couplée à la spectrométrie de masse tandem. Ces méthodes ont été utilisées pour le TDM des ARVs et pour les agents anti-VHC chez les patients infectés par le VIH, et le VHC, respectivement, mais aussi chez les patients co-infectés par le VIH-VHC. Un modèle de pharmacocinétique de population a été développé pour caractériser la variabilité inter-et intra-patient du médicament rilpivirine, un inhibiteur non nucléosidique de la transcriptase de VIH et d'identifier les variables génétiques et non génétiques influençant l'exposition au médicament. Aucun des facteurs étudiés n'a montré d'influence notable sur la clairance de la rilpivirine. Toutefois, la concentration résiduelle extrapolée selon le modèle de pharmacocinétique de population qui a été développé, a montré qu'une grande proportion des patients présente des concentrations minimales inférieures à la cible thérapeutique proposée. Dans ce contexte, la relation entre les concentrations minimales et l'échappement virologique nécessite une surveillance étroite des taux sanguins des patients recevant de la rilpivirine. A cet effet, le suivi thérapeutique est un outil important pour l'identification des patients à risque soient sous-exposés à lai rilpivirine. Pour identifier de nouveaux marqueurs de la toxicité qui pourraient induire l'arrêt du traitement, le profil des métabolites de phase I et de phase II a été étudié dans différentes matrices [plasma, LCR et urine) provenant de patients recevant de l'efavirenz. Les métabolites de phase II, qui n'avaient à ce jour jamais été investigués, constituent les principales espèces présentes dans les matrices étudiées. Au cours de ces investigations, un nouveau métabolite 8- OH-EFV-sulfate a été identifié chez l'homme, et ce dernier est. présent à des concentrations importantes. L'influence de certains facteurs pharmacogénétique des patients sur le profil des métabolites a été étudiée et ouvre la voie à de possibles nouveaux marqueurs phénotypiques alternatifs qui pourraient possiblement mieux prédire la toxicité associée au traitement par l'efavirenz. Finalement, nous nous sommes intéressés à étudier dans un modèle in vitro certains facteurs, comme la P-glycoprotéine, qui influencent la disposition cellulaire de certains médicaments antirétroviraux, en utilisant l'approche par la technologie du siRNA permettant de bloquer sélectivement l'expression du gène de cette protéine d'efflux des médicaments. -- Depuis l'introduction de la thérapie antiretrovirale (ARVs] la morbidité et la mortalité liées au VIH ont considérablement diminué. En parallèle le traitement contre le virus de l'hépatite C (VHC) a connu récemment d'énormes progrès avec l'arrivée de nouveaux médicaments puissants, ce qui a permis une augmentation considérable de la guérison de l'infection par le VHC. En dépit de l'efficacité de ces traitements antiviraux, les échecs thérapeutiques ainsi que les effets secondaires des traitements restent un problème important. Il a pu être démontré que la concentration de médicament présente dans l'organisme est corrélée avec l'efficacité clinique pour la plupart des médicaments agissant contre le VIH et contre le VHC. Les médicaments antiviraux sont généralement donnés à une posologie fixe et standardisée, à tous les patients, il existe cependant une importante variabilité entre les concentrations sanguines mesurées chez les individus. Cette variabilité peut être expliquée par plusieurs facteurs démographiques, environnementaux ou génétiques. Dans ce contexte, le suivi des concentrations sanguines (ou Therapeutic Drug Monitoring, TDM) permet de contrôler que les patients soient exposés à des concentrations suffisantes (pour bloquer la réplication du virus dans l'organisme) et éviter des concentrations excessives, ce qui peut entraîner l'apparition d'intolérence au traitement. Le but de ce travail de thèse est d'améliorer la compréhension des facteurs pharmacologiques et génétiques qui peuvent influencer l'efficacité et/ou la toxicité des médicaments antiviraux, dans le but d'améliorer le suivi des patients. A cet effet, des méthodes de dosage très sensibles et ont été mises au point pour permettre de quantifier les médicaments antiviraux dans le sang et dans d'autres liquides biologiques. Ces méthodes de dosage sont maintenant utilisées d'une part dans le cadre de la prise en charge des patients en routine et d'autre part pour diverses études cliniques chez les patients infectés soit par le HIV, le HCV ou bien coinfectés par les deux virus. Une partie de ce travail a été consacrée à l'investigation des différents facteurs démographiques, génétiques et environnementaux qui pourraient l'influencer la réponse clinique à la rilpivirine, un nouveau médicament contre le VIH. Toutefois, parmi tous les facteurs étudiés à ce jour, aucun n'a permis d'expliquer la variabilité de l'exposition à la rilpivirine chez les patients. On a pu cependant observer qu'à la posologie standard recommandée, un pourcentage relativement élevé de patients pourrait présenter des concentrations inférieures à la concentration sanguine minimale actuellement proposée. Il est donc utile de surveiller étroitement les concentrations de rilpivirine chez les patients pour identifier sans délai ceux qui risquent d'être sous-exposés. Dans l'organisme, le médicament subit diverses transformations (métabolisme) par des enzymes, notamment dans le foie, il est transporté dans les cellules et tissus par des protéines qui modulent sa concentration au site de son action pharmacologique. A cet effet, différents composés (métabolites) produits dans l'organisme après l'administration d'efavirenz, un autre médicament anti-VIH, ont été étudiés. En conclusion, nous nous sommes intéressés à la fois aux facteurs pharmacologiques et génétiques des traitements antiviraux, une approche qui s'inscrit dans l'optique d'une stratégie globale de prise en charge du patient. Dans ce contexte, le suivi des concentrations sanguines de médicaments constitue une des facettes du domaine émergent de la Médecine Personnalisée qui vise à maximiser le bénéfice thérapeutique et le profil de tolérance des médicaments antiviraux

Global transcriptional programs in peripheral nerve endoneurium and DRG are resistant to the onset of type 1 diabetic neuropathy in Ins2 mice.

While the morphological and electrophysiological changes underlying diabetic peripheral neuropathy (DPN) are relatively well described, the involved molecular mechanisms remain poorly understood. In this study, we investigated whether phenotypic changes associated with early DPN are correlated with transcriptional alterations in the neuronal (dorsal root ganglia [DRG]) or the glial (endoneurium) compartments of the peripheral nerve. We used Ins2(Akita/+) mice to study transcriptional changes underlying the onset of DPN in type 1 diabetes mellitus (DM). Weight, blood glucose and motor nerve conduction velocity (MNCV) were measured in Ins2(Akita/+) and control mice during the first three months of life in order to determine the onset of DPN. Based on this phenotypic characterization, we performed gene expression profiling using sciatic nerve endoneurium and DRG isolated from pre-symptomatic and early symptomatic Ins2(Akita/+) mice and sex-matched littermate controls. Our phenotypic analysis of Ins2(Akita/+) mice revealed that DPN, as measured by reduced MNCV, is detectable in affected animals already one week after the onset of hyperglycemia. Surprisingly, the onset of DPN was not associated with any major persistent changes in gene expression profiles in either sciatic nerve endoneurium or DRG. Our data thus demonstrated that the transcriptional programs in both endoneurial and neuronal compartments of the peripheral nerve are relatively resistant to the onset of hyperglycemia and hypoinsulinemia suggesting that either minor transcriptional alterations or changes on the proteomic level are responsible for the functional deficits associated with the onset of DPN in type 1 DM.

Towards a global criteria based framework for the sustainability assessment of bioethanol supply chains: application to the Swiss dilemma : is local produced bioethanol more sustainable than bioethanol imported from Brazil?

Biofuels are considered as a promising substitute for fossil fuels when considering the possible reduction of greenhouse gases emissions. However limiting their impacts on potential benefits for reducing climate change is shortsighted. Global sustainability assessments are necessary to determine the sustainability of supply chains. We propose a new global criterion based framework enabling a comprehensive international comparison of bioethanol supply chains. The interest of this framework is that the selection of the sustainability indicators is qualified on three criterions: relevance, reliability and adaptability to the local context. Sustainability issues have been handled along environmental, social and economical issues. This new framework has been applied for a specific issue: from a Swiss perspective, is locally produced bioethanol in Switzerland more sustainable than imported from Brazil? Thanks to this framework integrating local context in its indicator definition, Brazilian production of bioethanol is shown as energy efficient and economically interesting for Brazil. From a strictly economic point of view, bioethanol production within Switzerland is not justified for Swiss consumption and questionable for the environmental issue. The social dimension is delicate to assess due to the lack of reliable data and is strongly linked to the agricultural policy in both countries. There is a need of establishing minimum sustainability criteria for imported bioethanol to avoid unwanted negative or leakage effects.

Multicopy suppression of a gacA mutation by the infC operon in Pseudomonas fluorescens CHA0: competition with the global translational regulator RsmA.

The gacA gene of the biocontrol strain Pseudomonas fluorescens CHA0 codes for a response regulator which, together with the sensor kinase GacS (=LemA), is required for the production of exoenzymes and secondary metabolites involved in biocontrol, including hydrogen cyanide (HCN). A gacA multicopy suppressor was isolated from a cosmid library of strain CHA0 and identified as the infC-rpmI-rplT operon, which encodes the translation initiation factor IF3 and the ribosomal proteins L35 and L20. The efficiency of suppression was about 30%, as determined by the use of a GacA-controlled reporter construct, i.e. a translational hcnA'-'lacZ fusion. Overexpression of the rsmA gene (coding for a global translational repressor) reversed the suppressive effect of the amplified infC operon. This finding suggests that some product(s) of the infC operon can compete with RsmA at the level of translation in P. fluorescens CHA0 and that important biocontrol traits can be regulated at this level.