Vers une meilleure compréhension de la pathogenèse de la maladie osseuse reliée à la fibrose kystique

Les patients atteints de la fibrose kystique (FK) ont désormais un âge médian de survie dépassant la cinquantaine. Par contre, avec ce vieillissement surviennent de nouvelles complications dont l'une des plus prévalente est la maladie osseuse associée à la FK. Les souris dont le Cftr est invalidé génétiquement présentent une densité osseuse amoindrie qui découle d’un débalancement du remodelage osseux caractérisé par une diminution de la formation et une augmentation de la résorption osseuse. L'observation que plusieurs modèles murins FK ont un phénotype ostéopénique et ce, même en absence de certains facteurs étiologiques (inflammation chronique, prise de glucocorticoïdes, insuffisance pancréatique etc.) laisse croire que le Cftr, le gène muté dans la FK, joue un rôle non-négligeable dans le métabolisme osseux. Le présent projet étudiera l’impact de l’absence du CFTR, sur les ostéoblastes (Ob) et ostéoclastes (Oc) dans un modèle murin de FK, soit les souris Cftr-/- de souche BALB/c. De plus, les Ob, sont reconnus comme ayant un effet modulateur sur le microenvironnement leucocytaire de la moelle osseuse (MO). Ce projet visera également à investiguer l’impact de l’absence du CFTR sur la niche leucocytaire de la MO. Nos résultats de densitométrie osseuse et de microtomographie à rayons X ont confirmé que les souris Cftr -/- ont une densité osseuse et un contenu minéral osseux abaissé, une diminution du volume osseux trabéculaire, un nombre amoindri de travées osseuses et une plus grande séparation entre les travées comparé aux souris Cftr+/+. Afin de mieux comprendre ce phénotype osseux, nous avons vérifié et confirmé que l’expression génique et protéique du CFTR est présente chez des Ob dérivés de la MO, mais est absent au niveau des Oc dérivés de la MO. Ces observations corroborent nos résultats portant sur la différenciation des cellules osseuses où nous avons démontré que seule la différenciation et fonction ostéoblastique sont affectées par l'absence du CFTR. Ce défaut ostéoblastique semble influencer négativement la leucopoïèse puisque nous observons une quantité moindre de cellules T, de macrophages et de cellules dendritiques chez les souris Cftr -/- vs. Cftr +/+. À la lumière de ces résultats, l'absence du CFTR semble avoir un impact important sur les ostéoblastes et la moelle osseuse.

Rôle du facteur de transcription Gfi1b au cours de l’hématopoïèse précoce

Le facteur de transcription Growth factor independent 1b (GFI1b) est impliqué à différents stades dans la régulation de l'hématopoïèse. Il est notamment fortement exprimé dans les cellules souches hématopoïétiques et au cours de la différenciation des cellules des lignées érythroïdes et mégacaryocytaires. Grâce à un modèle de délétion conditionnelle chez la souris par le système CreLox, nous avons montré que l'absence de GFI1b entraîne une prolifération de cellules mégacaryocytaires incapables de produire des plaquettes. Notre étude ne permet pas de confirmer formellement la prolifération des cellules souches dans la moelle osseuse précédemment observée par notre équipe dans un autre modèle murin. En revanche, les souris GFI1b "knockout" présentent une augmentation des cellules souches circulantes dans le sang périphérique. Une analyse moléculaire préliminaire montre que GFI1b pourrait influer sur la régulation par le cycle circadien de la mobilisation de ces cellules dans le sang. Finalement, notre étude des effets biologiques de la délétion de GFI1b dans le compartiment hématopoïétique des souris adultes nous a permis de définir de façon plus précise le rôle de GFI1b dans l'hématopoïèse et de confirmer son rôle majeur dans la régulation de la mégacaryopoïèse et la production de plaquettes matures.

Les immunoglobulines intraveineuses et la réponse spécifique des cellules T dans la prévention de la maladie lymphoproliférative post-greffe associée au virus Epstein-Barr chez les enfants greffés de cellules souches hématopoïétiques.

La maladie lymphoproliférative post-greffe (MLP) est une complication grave chez les greffés (d’organes solides ou de cellules souches hématopoïétiques) immunosupprimés suite à l'infection par le virus Epstein-Barr (VEB). En l’absence d'une réponse efficace des lymphocytes T cytotoxiques, les cellules B infectées par le VEB peuvent proliférer et donner lieu à la MLP. Dans le cas des receveurs de greffe immunosupprimés, les cellules B infectées par le VEB de façon lytique, produisent activement de nouveaux virions. Ces derniers infectent les cellules B voisines, entraînant leur expansion polyclonale. La gp350, une protéine du cycle lytique située dans l'enveloppe virale, joue un rôle important dans l'infection par le VEB. Elle interagit avec le récepteur CD21 exprimée à la surface des cellules B pour permettre l’entrée du virus. Ainsi, des anticorps neutralisants anti-gp350 sont considérés être des acteurs clés dans le blocage de l'infection, empêchant ainsi le développement de la MLP. L'effet protecteur des immunoglobulines intraveineuses (IgIV) à titre prophylactique contre le VEB et la MLP chez les greffés de cellules souches hématopoïétiques n’est pas clairement démontré. Par conséquent, le premier objectif de cette thèse a proposé d'évaluer l'efficacité des IgIV contre l'infection par le VEB et la MLP chez les receveurs de cellules souches hématopoïétiques. Le deuxième objectif a proposé de déterminer, en utilisant la technique ELISpot, si la présence d'une réponse forte des lymphocytes T contre l'antigène précoce BMLF1 du cycle lytique du VEB pourrait constituer un marqueur de protection contre la MLP chez les greffés de cellules souches hématopoïétiques. Les résultats ont montré d'une part que, si les IgIV peuvent neutraliser efficacement l'infection par le VEB in vitro, ils ne protègent pas efficacement les patients greffés contre l'infection par le VEB in vivo. D'autre part, l'étude de la réponse des lymphocytes T contre des antigènes du VEB a démontré que les cellules T de certains patients sont capables de reconnaître l'antigène lytique BMLF1. Cette réponse spécifique des lymphocytes T peut s’avérer un bon marqueur de la protection contre la MLP. Les résultats de cette thèse démontrent que l’infection lytique au VEB joue un rôle fondamental dans le développement de la MLP. Les données indiquent également que la présence d'une réponse spécifique des lymphocytes T contre un antigène du cycle lytique du VEB peut constituer un bon marqueur de la protection contre la MLP. Cependant, le traitement des patients recevant des greffes de cellules souches hématopoïétiques avec les IgIV n’apparaît pas efficace dans la prévention de la MLP.

Régulation de l'expression de PPARγ dans l'arthrose

L’arthrose (OA) est une maladie dégénérative très répondue touchant les articulations. Elle est caractérisée par la destruction progressive du cartilage articulaire, l’inflammation de la membrane synoviale et le remodelage de l’os sous chondral. L’étiologie de cette maladie n’est pas encore bien définie. Plusieurs études ont été menées pour élucider les mécanismes moléculaires et cellulaires impliqués dans le développement de l’OA. Les effets protecteurs du récepteur activé par les proliférateurs de peroxysomes gamma (PPARγ) dans l'OA sont bien documentés. Il a été démontré que PPARγ possède des propriétés anti-inflammatoires et anti-cataboliques. Aussi, plusieurs stimuli ont été impliqués dans la régulation de l’expression de PPARγ dans différents types cellulaires. Cependant, les mécanismes exacts responsables de cette régulation ainsi que le profil de l’expression de ce récepteur au cours de la progression de l’OA ne sont pas bien connus. Dans la première partie de nos travaux, nous avons essayé d’élucider les mécanismes impliqués dans l’altération de l’expression de PPARγ dans cette maladie. Nos résultats ont confirmé l’implication de l’interleukine-1β (IL-1β), une cytokine pro-inflammatoire, dans la réduction de l’expression de PPARγ au niveau des chondrocytes du cartilage articulaire. Cet effet coïncide avec l'induction de l’expression du facteur de transcription à réponse précoce de type 1 (Egr-1). En plus, la diminution de l'expression de PPARγ a été associée au recrutement d'Egr-1 et la réduction concomitante de la liaison de Sp1 au niveau du promoteur de PPARγ. Dans la deuxième partie de nos travaux, nous avons évalué le profil d’expression de ce récepteur dans le cartilage au cours de la progression de cette maladie. Le cochon d’inde avec OA spontanée et le chien avec OA induite par rupture du ligament croisé antérieur (ACLT) deux modèles animaux d’OA ont été utilisés pour suivre l’expression des trois isoformes de PPARs : PPAR alpha (α), PPAR béta (β) et PPAR gamma (γ) ainsi que la prostaglandine D synthase hématopoïétique (H-PGDS) et la prostaglandine D synthase de type lipocaline (L-PGDS) deux enzymes impliquées dans la production de l’agoniste naturel de PPARγ, la 15-Deoxy-delta(12,14)-prostaglandine J(2) (15d-PGJ2). Nos résultats ont démontré des changements dans l’expression de PPARγ et la L-PGDS. En revanche, l’expression de PPARα, PPARβ et H-PGDS est restée stable au fil du temps. La diminution de l’expression de PPARγ dans le cartilage articulaire semble contribuer au développement de l’OA dans les deux modèles animaux. En effet, le traitement des chondrocytes par de siRNA dirigé contre PPARγ a favorisé la production des médiateurs arthrosiques tels que l'oxyde nitrique (NO) et la métalloprotéase matricielle de type 13 (MMP-13), confirmant ainsi le rôle anti-arthrosique de ce récepteur. Contrairement à ce dernier, le niveau d'expression de la L-PGDS a augmenté au cours de la progression de cette maladie. La surexpression de la L-PGDS au niveau des chondrocytes humains a été associée à la diminution de la production de ces médiateurs arthrosiques, suggérant son implication dans un processus de tentative de réparation. En conclusion, l’ensemble de nos résultats suggèrent que la modulation du niveau d’expression de PPARγ, de la L-PGDS et d’Egr-1 au niveau du cartilage articulaire pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et probablement d’autres formes d'arthrite.

Mécanismes d'action des cellules stromales mésenchymateuses dans le traitement de la réaction du greffon contre l'hôte

La maladie du greffon contre l’hôte (GvHD) est un effet secondaire sérieux de la transplantation de cellules souches hématopoïétiques (HSCT). Cette maladie entraine une haute mortalité et ses symptômes sont dévastateurs. Les traitements actuels de la GvHD comportent plusieurs produits, tels les corticostéroïdes, mais ces derniers sont immunosuppresseurs et leurs effets secondaires sont aussi très dommageables pour les patients et leur guérison. Les cellules stromales mésenchymateuses (MSC) représentent une alternative ou une addition potentielle de traitement pour la GvHD et ces cellules ne semblent pas posséder les effets secondaires des traitements classiques. Un nombre important d’études cliniques faisant l’objet des MSC ont été enregistrées. Malgré cet engouement, le mécanisme de leur immunomodulation reste encore à élucider. Notre objectif est donc de mieux définir ce mécanisme. Nous avons utilisé un modèle simplifié pour simuler la GvHD in vitro. Ce modèle se base sur la stimulation de lymphocytes CD4+ par des cellules dendritiques allogéniques. La mesure de la prolifération de ces cellules stimulées sert d’indicateur de leur réactivité. Selon les résultats obtenus par la technologie CRISPR de génie génétique, les MSC exerceraient leur immunosuppression sur les cellules T CD4+ principalement par la sécrétion de l’enzyme IDO1. Les MSC seraient également capables d’induire certaines cellules CD4+ en cellules régulatrices, un processus indépendant de la sécrétion d’IDO1. Toutefois, ces cellules ne semblent pas correspondre aux cellules Treg conventionnelles.

Les oncogènes NUP98-PHF23 et NUP98-HOXD13 confèrent un potentiel aberrant d’auto-renouvellement aux progéniteurs thymiques.

L’initiation de la leucémogénèse dans la leucémie aigue lymphoblastique (LAL)-T résulte de l’activation aberrante de facteurs de transcription de la lignée lymphocytaire T. Nous démontrons que les gènes de fusion NUP98-PHF23 (NP23) et NUP98-HOXD13 (NHD13) reprogramment les thymocytes normaux en cellules souches pré-leucémiques (CS-préL) possédant un potentiel aberrant d’auto-renouvellement. Basé sur des essais de clonalité performés sur des thymocytes transplantés en série, nous avons découvert que cette population est hiérarchisée similairement aux cellules souches hématopoïétiques normales. Ces CS-préL dévoilent un enrichissement du compartiment de précurseurs thymiques immatures KIT+ où les deux oncogènes, NP23 et NHD13, activent des gènes impliqués dans l’autorenouvellement, incluant Hoxa9, Hoxa10, Lyl1 et Hhex. De plus, l’activité d’autorenouvellement est abrogée par les ARN interférents contre Lyl1 et Hhex, indiquant leur implication fonctionnelle en aval de NP23 et NHD13. Puisque ces gènes sont aussi activés en aval de trois autres oncogènes dans la LAL-T, SCL/TAL1, LMO1 et LMO2, nous concluons que les niveaux d’activation de Lyl1 et Hhex fixent le seuil de reprogrammation des thymocytes normaux en CS-préL. Malgré l'efficacité des traitements de chimiothérapie actuels à diminuer la masse tumorale, les CS-préL sont épargnées, pouvant mener à des rechutes. Nos résultats répondent à ce besoin et proposent de nouvelles avenues permettant de cibler les CS-préL du compartiment de thymocytes immatures dans la LAL-T.

Purification and characterisation of Vibriophage isolated from Mangalavanam mangrove

The temperate, filamentous phage ФMV -5 isolated from Mangalavanam mangrove of Kochi, using the environmental strain of Vibrio sp. MV-5 shares many similar properties with other marine phage isolates, while also remaining unique. The study has revealed that the interaction of temperate phages and the microbial population in the marine environment may contribute significantly to microbial genetic diversity and composition by conversion and transduction and which requires greater study.Prophages contribute a substantial share of the mobile DNA of their bacterial hosts and seem to influence the short-term evolution of pathogenic bacteria. Automated methods for systematic investigation of prophages and other mobile DNA elements in the available bacterial genome sequences will be necessary to understand their role in bacterial genome evolution. In the past, phages were mainly investigated as the simplest model systems in molecular biology. Now it is increasingly realized that phage research will be instrumental in the understanding of bacterial abundance in the environment. One can predict that phage research will impact diverse areas such as geochemistry and medicine. Success will largely depend on integrative multidisciplinary approaches in this field. Clearly, further studies are required to understand how vibriophages interact with Vibrios to promote this organism's acquisition of the critical genes which alter its virulence or adaptation to its environmental niche.It is evident from this study and comparison with those reports cited above that vibriophage ФMV-5 is a previously unreported bacteriophage. It is recommended that the minimum requirement for reporting a new phage should be novel morphological markers and a description of host range, both of which have been achieved in this study.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

Vibrio are important during hatchery rearing. aquaculture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp farmers and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases.With the progress in aquaculture, intensive systems used for shrimp aquaculture create an artificial environment that increases bacterial growth. To maintain the productivity of such an intensive aquaculture, high inputs of fish protein have to be employed for feeding together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence. The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose, water, feed, shrimp quality and aquaculture management.Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio.Of the l2 pathogenic Vibrio species, 8 species are known to be directly food associated. Strict quality guidelines have been laid by the importing nations, for the food products that enter their markets. The microbiological quality requirement for export of frozen shrimp products is that V.cholerae, V.parahaemolyticus and V. vulnificus should be absent in 25g of the processed shrimp (Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic vibrios, the shrimp aquaculture system.So the present study was undertaken to study the incidence of pathogenic Vibrio spp. in Penaeus monodon shrimp hatcheries and aquaculture farms, to carry out biochemical investigations of the pathogenic Vibrio spp isolated from P. monodon hatchery and. aquaculture environments, to assess the effect of salt (NaCl) on the growth and enzymatic activities of pathogenic Vibrio spp., to study the effect of preservatives, and chemicals on the growth of pathogenic Vibrio spp. and to employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.Samples of water (n=7) and post-larvae (n=7) were obtained from seven Penaeus monodon hatcheries and samples of water (n=5), sediment (n=5) and shrimp (n=5) were obtained from five P. monodon aquaculture farms located on the East Coast of lndia. The microbiological examination of water, sediment, post-larvae and shrimp samples was carried out employing standard methods and by using standard media.The higher bacterial loads were obtained in pond sediments which can be attributed to the accumulation of organic matter at the pond bottom which stimulated bacterial growth.Shrimp head. (4.78 x 105 +/- 3.0 x 104 cfu/g) had relatively higher bacterial load when compared to shrimp muscle 2.7 x 105 +/- 1.95 x 104 cfu/g). ln shrimp hatchery samples, the post-larvae (2.2 x 106 +/- 1.9 x 106 cfu/g) had higher bacterial load than water (5.6 x 103 +/- 3890 cfu/ml).The mean E.coli counts were higher in aquaculture pond sediment (204+/-13 cfu/g) and pond water (124+/-88 cfu/ml). Relatively lower Escherichia coli counts were obtained from shrimp samples (12+/-11 to 16+/-16.7 cfu/g). The presence of E.coli in aquaculture environment might have been from the source water. E.coli was not detected in hatchery waters and post-larvae.

Influence of personality factors on the consumption of personal care products

The study is significant from both an application perspective of marketing management as well as from an academic angle. The market for personal care products is a highly fragmented one, with intense competition for specific niche segments. It is well known in marketing literature that the bulk of the volume of sale is accounted for by the minority who are the heavy users. This study will help the marketers to identify the personality profile of such a group and understand how the interaction of personality factors at least partially explains differences in consumption. This knowledge might be useful for better segmentation using psychographic variables as well as for designing specific advertisement campaigns to target the vulnerable groups of customers. From a theoretical perspective, the research may contribute to understanding how specific personality variables and their interaction lead to differences in consumption. The knowledge corresponding to self theory, social comparison theory, persuasibility, evidence from psychology of eating disorders: these all may be integrated into a common frame work for explaining consumption of products having a social function.

Macrobenthos And Its Relation To Ecosystem Dynamics In The Cochin Estuary

Cochin estuary is a shallow brackish water body situated on the south west coast of India. It is a tropical positive estuary extending between 90 40’ and 100 12’ N and 760 10’and 760 30’ E with its northern boundary at Azhikode and southern boundary at Thannermukkom bund.The abundance of benthic fauna in an ecosystem shows the close relationship to its environment and reflects the characteristics of an ecological niche. Seasonal and monthly variations in the distribution of macrobenthos in relation to sediment characteristics were conducted in Cochin estuary from 2009-10 periods. Oxidation-reduction potential showed reducing trends that affected the distribution and diversity of fauna. Seasonal variations in water quality and river discharge pattern affected the faunal composition in the different stations. Sewage mixing was the principal source of organic pollution in the Cochin estuary. The sediment pH was generally on the alkaline side ranging from 4.99 at St.9 and 8.33 at St.1.The Eh ranged from -11mV at St.3 to -625mV at St.2.The temperature varied from 260C to 320C in the estuary. The moisture content ranged from 1.63 to 12.155%, that of organic carbon from 0 09 at St. 6 to 4.29% at St.9 and that of organic matter from 0.16 to 7.39%. Seasonally, the average of Eh was highest during the monsoon (156.22 mV) and in the pre monsoon (140.94 mV). The average pH for the 9 study stations was 7.68 during monsoon period and 7.08 during post monsoon. Based on group wise seasonal analysis, the average mean abundance was maximum for polychaetes (43.47) followed by nematodes (33.62), crustaceans (21.62), molluscs (11.94) and Pisces (0.05) in the estuary. Monsoon season was most favourable for benthic faunal abundance followed by the post monsoon period in the study. The series of human interventions like dredging, discharge of industrial effluents, urbanisation and related aspects had a strong influence on the distribution, abundance of benthic macrofauna in the wetland.

Differential induction, isolation, physicochemical and molecular characterization of temperate phages of environmental Vibrio cholerae as evidence of phage mediated Horizontal Gene Transfer

The resurgence of the enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries like India. The southern Indian state of Kerala is endemic to cholera. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. Marine aquaculture settings and mangrove environments of Kerala serve as reservoirs for V. cholerae. The non-O1/non-O139 environmental isolates of V. cholerae with incomplete ‘virulence casette’ are to be dealt with caution as they constitute a major reservoir of diverse virulence genes in the marine environment and play a crucial role in pathogenicity and horizontal gene transfer. The genes coding cholera toxin are borne on, and can be infectiously transmitted by CTXΦ, a filamentous lysogenic vibriophages. Temperate phages can provide crucial virulence and fitness factors affecting cell metabolism, bacterial adhesion, colonization, immunity, antibiotic resistance and serum resistance. The present study was an attempt to screen the marine environments like aquafarms and mangroves of coastal areas of Alappuzha and Cochin, Kerala for the presence of lysogenic V. cholerae, to study their pathogenicity and also gene transfer potential. Phenotypic and molecular methods were used for identification of isolates as V. cholerae. The thirty one isolates which were Gram negative, oxidase positive, fermentative, with or without gas production on MOF media and which showed yellow coloured colonies on TCBS (Thiosulfate Citrate Bile salt Sucrose) agar were segregated as vibrios. Twenty two environmental V. cholerae strains of both O1 and non- O1/non-O139 serogroups on induction with mitomycin C showed the presence of lysogenic phages. They produced characteristic turbid plaques in double agar overlay assay using the indicator strain V. cholerae El Tor MAK 757. PCR based molecular typing with primers targeting specific conserved sequences in the bacterial genome, demonstrated genetic diversity among these lysogen containing non-O1 V. cholerae . Polymerase chain reaction was also employed as a rapid screening method to verify the presence of 9 virulence genes namely, ctxA, ctxB, ace, hlyA, toxR, zot,tcpA, ninT and nanH, using gene specific primers. The presence of tcpA gene in ALPVC3 was alarming, as it indicates the possibility of an epidemic by accepting the cholera. Differential induction studies used ΦALPVC3, ΦALPVC11, ΦALPVC12 and ΦEKM14, underlining the possibility of prophage induction in natural ecosystems, due to abiotic factors like antibiotics, pollutants, temperature and UV. The efficiency of induction of prophages varied considerably in response to the different induction agents. The growth curve of lysogenic V. cholerae used in the study drastically varied in the presence of strong prophage inducers like antibiotics and UV. Bacterial cell lysis was directly proportional to increase in phage number due to induction. Morphological characterization of vibriophages by Transmission Electron Microscopy revealed hexagonal heads for all the four phages. Vibriophage ΦALPVC3 exhibited isometric and contractile tails characteristic of family Myoviridae, while phages ΦALPVC11 and ΦALPVC12 demonstrated the typical hexagonal head and non-contractile tail of family Siphoviridae. ΦEKM14, the podophage was distinguished by short non-contractile tail and icosahedral head. This work demonstrated that environmental parameters can influence the viability and cell adsorption rates of V. cholerae phages. Adsorption studies showed 100% adsorption of ΦALPVC3 ΦALPVC11, ΦALPVC12 and ΦEKM14 after 25, 30, 40 and 35 minutes respectively. Exposure to high temperatures ranging from 50ºC to 100ºC drastically reduced phage viability. The optimum concentration of NaCl required for survival of vibriophages except ΦEKM14 was 0.5 M and that for ΦEKM14 was 1M NaCl. Survival of phage particles was maximum at pH 7-8. V. cholerae is assumed to have existed long before their human host and so the pathogenic clones may have evolved from aquatic forms which later colonized the human intestine by progressive acquisition of genes. This is supported by the fact that the vast majority of V. cholerae strains are still part of the natural aquatic environment. CTXΦ has played a critical role in the evolution of the pathogenicity of V. cholerae as it can transmit the ctxAB gene. The unusual transformation of V. cholerae strains associated with epidemics and the emergence of V. cholera O139 demonstrates the evolutionary success of the organism in attaining greater fitness. Genetic changes in pathogenic V. cholerae constitute a natural process for developing immunity within an endemically infected population. The alternative hosts and lysogenic environmental V. cholerae strains may potentially act as cofactors in promoting cholera phage ‘‘blooms’’ within aquatic environments, thereby influencing transmission of phage sensitive, pathogenic V. cholerae strains by aquatic vehicles. Differential induction of the phages is a clear indication of the impact of environmental pollution and global changes on phage induction. The development of molecular biology techniques offered an accessible gateway for investigating the molecular events leading to genetic diversity in the marine environment. Using nucleic acids as targets, the methods of fingerprinting like ERIC PCR and BOX PCR, revealed that the marine environment harbours potentially pathogenic group of bacteria with genetic diversity. The distribution of virulence associated genes in the environmental isolates of V. cholerae provides tangible material for further investigation. Nucleotide and protein sequence analysis alongwith protein structure prediction aids in better understanding of the variation inalleles of same gene in different ecological niche and its impact on the protein structure for attaining greater fitness of pathogens. The evidences of the co-evolution of virulence genes in toxigenic V. cholerae O1 from different lineages of environmental non-O1 strains is alarming. Transduction studies would indicate that the phenomenon of acquisition of these virulence genes by lateral gene transfer, although rare, is not quite uncommon amongst non-O1/non-O139 V. cholerae and it has a key role in diversification. All these considerations justify the need for an integrated approach towards the development of an effective surveillance system to monitor evolution of V. cholerae strains with epidemic potential. Results presented in this study, if considered together with the mechanism proposed as above, would strongly suggest that the bacteriophage also intervenes as a variable in shaping the cholera bacterium, which cannot be ignored and hinting at imminent future epidemics.

The role of Ken&Barbie in JAK/STAT signalling during development of Drosophila melanogaster

Cell-cell interactions during embryonic development are crucial in the co-ordination of growth, differentiation and maintenance of many different cell types. To achieve this co-ordination each cell must properly translate signals received from neighbouring cells, into spatially and temporally appropriate developmental responses. A surprisingly limited number of signal pathways are responsible for the differentiation of enormous variety of cell types. As a result, pathways are frequently 'reused' during development. Thus, in mammals the JAK/STAT pathway is required during early embryogenesis, mammary gland formation, hematopoiesis and, finally, plays a pivotal role in immune response. In the canonical way, the JAK/STAT pathway is represented by a transmembrane receptor associated with a Janus kinase (JAK), which upon stimulation by an extra-cellular ligand, phosphorylates itself, the receptor and, finally, the signal transducer and activator of transcription (STAT) molecules. Phosphorylated STATs dimerise and translocate to the nucleus where they activate transcription of target genes. The JAK/STAT pathway has been conserved throughout evolution, and all known components are present in the genome of Drosophila melanogaster. Besides hematopoietic and immunity functions, the pathway is also required during development for processes including embryonic segmentation, tracheal morphogenesis, posterior spiracle formation etc. This study describes Drosophila Ken&Barbie (Ken) as a selective regulator of JAK/STAT signalling. ken mutations identified in a screen for modulators of an eye overgrowth phenotype, caused by over-expression of the pathway ligand unpaired, also interact genetically with the pathway receptor domeless (dome) and the transcription factor stat92E. Over-expression of Ken can phenocopy developmental defects known to be caused by the loss of JAK/STAT signalling. These genetic interactions suggest that Ken may function as a negative regulator of the pathway. Ken has C-terminal Zn-finger domain, presumably for DNA binding, and N-terminal BTB/POZ domain, often found in transcriptional repressors. Using EGFP-fused construct expressed in vivo revealed nuclear accumulation of Ken. Therefore, it is proposed that Ken may act as a suppresser of STAT92E target genes. An in vitro assay, termed SELEX, determined that Ken specifically binds to a DNA sequence, with the essential for DNA recognition core overlapping that of STAT92E. This interesting observation suggests that not all STAT92E sites may also allow Ken binding. Strikingly, when effects of ectopic Ken on the expression of putative JAK/STAT pathway target genes were examined, only a subset of the genes tested, namely vvl, trh and kni, were down-regulated by Ken, whereas some others, such as eve and fj, appeared to be unresponsive. Further analysis of vvl, one of the genes susceptible to ectopic Ken, was undertaken. In the developing hindgut, expression of vvl is JAK/STAT pathway dependent, but remains repressed in the posterior spiracles, despite the stimulation of STAT92E by Upd in their primordia. Importantly, ken is also expressed in the developing posterior spiracles. Strikingly, up-regulation of vvl is observed in these tissues in ken mutant embryos. These imply that while ectopic Ken is sufficient to repress the expression of vvl in the hindgut, endogenous Ken is also necessary to prevent its activation in the posterior spiracles. It is therefore conceivable that ectopic vvl expression in the posterior spiracles of the ken mutants may be the result of de-repression of endogenous STAT92E activity. Another consequence of these observations is a fine balance that must exist between STAT92E and Ken activities. Apparently, endogenous level of Ken is sufficient to repress vvl, but not other, as yet unidentified, JAK/STAT pathway targets, whose presumable activation by STAT92E is required for posterior spiracle development as the embryos mutant for dome, the receptor of the pathway, show severe spiracle defects. These defects are also observed in the embryos mis-expressing Ken. Though it is possible that the posterior spiracle phenotype caused by higher levels of Ken results from a JAK/STAT pathway independent activity, it seems to be more likely that Ken acts in a dosage dependent manner, and extra Ken is able to further antagonise JAK/STAT pathway target genes. While STAT92E binding sites required for target gene expression have been poorly characterised, the existence of genome data allows the prediction of candidate STAT92E sites present in target genes promoters to be attempted. When a 6kb region containing the putative regulatory domains flanking the vvl locus are examined, only a single potential STAT92E binding site located 825bp upstream of the translational start can be detected. Strikingly, this site also includes a perfect Ken binding sequence. Such an in silico observation, though consistent with both Ken DNA binding assay in vitro and regulation of STAT92E target genes in vivo, however, requires further analysis. The JAK/STAT pathway is implicated in a variety of processes during embryonic and larval development as well as in imago. In each case, stimulation of the same transcription factor results in different developmental outcomes. While many potential mechanisms have been proposed and demonstrated to explain such pleiotropy, the present study indicates that Ken may represent another mechanism, with which signal transduction pathways are controlled. Ken selectively down-regulates a subset of potential target genes and so modifies the transcriptional profile generated by activated STAT92E - a mechanism, which may be partially responsible for differences in the morphogenetic processes elicited by JAK/STAT signalling during development.

A User-Centric Perspective on Parallel Programming with Focus on OpenMP

The process of developing software that takes advantage of multiple processors is commonly referred to as parallel programming. For various reasons, this process is much harder than the sequential case. For decades, parallel programming has been a problem for a small niche only: engineers working on parallelizing mostly numerical applications in High Performance Computing. This has changed with the advent of multi-core processors in mainstream computer architectures. Parallel programming in our days becomes a problem for a much larger group of developers. The main objective of this thesis was to find ways to make parallel programming easier for them. Different aims were identified in order to reach the objective: research the state of the art of parallel programming today, improve the education of software developers about the topic, and provide programmers with powerful abstractions to make their work easier. To reach these aims, several key steps were taken. To start with, a survey was conducted among parallel programmers to find out about the state of the art. More than 250 people participated, yielding results about the parallel programming systems and languages in use, as well as about common problems with these systems. Furthermore, a study was conducted in university classes on parallel programming. It resulted in a list of frequently made mistakes that were analyzed and used to create a programmers' checklist to avoid them in the future. For programmers' education, an online resource was setup to collect experiences and knowledge in the field of parallel programming - called the Parawiki. Another key step in this direction was the creation of the Thinking Parallel weblog, where more than 50.000 readers to date have read essays on the topic. For the third aim (powerful abstractions), it was decided to concentrate on one parallel programming system: OpenMP. Its ease of use and high level of abstraction were the most important reasons for this decision. Two different research directions were pursued. The first one resulted in a parallel library called AthenaMP. It contains so-called generic components, derived from design patterns for parallel programming. These include functionality to enhance the locks provided by OpenMP, to perform operations on large amounts of data (data-parallel programming), and to enable the implementation of irregular algorithms using task pools. AthenaMP itself serves a triple role: the components are well-documented and can be used directly in programs, it enables developers to study the source code and learn from it, and it is possible for compiler writers to use it as a testing ground for their OpenMP compilers. The second research direction was targeted at changing the OpenMP specification to make the system more powerful. The main contributions here were a proposal to enable thread-cancellation and a proposal to avoid busy waiting. Both were implemented in a research compiler, shown to be useful in example applications, and proposed to the OpenMP Language Committee.

Nachhaltige Energie für ländliche Entwicklung in dem Sub-Sahara Afrika: interdisziplinäre Herangehensweise und organisatorische Herausforderung

Angesichts der Geschichte der Entwicklungspolitik, ist diese Arbeit darauf ausgerichtet, einige Beobachtungen in Bezug auf die so genannte Entwicklung hervorzuheben; insbesondere auf die andauernde prekäre Situation und Armut in ländlichen afrikanischen Gebieten. Armut ist nach Amartya SEN – weiter präzisiert von J.L. Dubois – die Deprivation von „Fähigkeiten“, die Individuen und lokale Gemeinschaften zu ausgeschlossenen und vergessenen Akteuren des Systems machen. Das nennt Paulo Freire, das Menschen zu „Objekten“ gemacht werden. Es rechtfertigt die starke Annahme, die in dieser Studie getroffen wird, dass vielmehr die Menschen als „Subjekte“ ihrer Veränderung und Entwicklung im Mittelpunkt stehen. Die Arbeit zeigt und erklärt in historischer Chronologie, wie die Entwicklungspolitiken und unterschiedliche Beteiligte auf allen Ebenen diese Situation verursachen. Trotz alledem bleiben die Individuen und lokalen Gemeinschaften, die in Symbiose mit ihrer natürlichen Umwelt leben, die reich an verschiedenen Ressourcen und Potentialen ist, als Reaktion darauf und gleichzeitig als Überlebensstrategie zutiefst verbunden mit dem, was sie vor Ort haben, womit sie eine tiefere und intensive Beziehung besitzen, wenn man von ihrer Geschichte, ihrer Kultur und der Handlungslogik ausgeht. Für externe Akteure, die sie über das vorhandene System dominieren und beeinflussen bleiben sie „Objekte“, aber in der Vielzahl ihrer endogenen Initiativen, zeigen sie die Fähigkeit und Substanz, die beweisen, dass sie auf ihrer Ebene das eigentliche Subjekt sind, die dynamischen Akteure. Aber isolierte Initiativen auf spezifische reale Bedürfnisse bei gleichzeitiger Dominierung durch das System mit seiner Marktlogik, führt dies langfristig nur zu dem Zirkulus Vitiosus der Armut. Daher ist eine ganzheitliche Sicht entscheidend für nachhaltige Entwicklung und für die notwendige Veränderung. Es geht nicht nur um die Veränderung des Systems und die Wahl politischer Maßnahmen, sondern genau genommen um das Verhalten der Akteure auf allen Ebenen und die Art der Beziehungen zwischen ihnen allen. Es ist eine Frage des erneuten Überdenkens des Entwicklungspfades, der andere Logik, Visionen, Interessen und Strategien aller Beteiligten, unserer so genannten Akteure einschließt. Ob dies von endogenen Initiativen oder neuen gemeinsamen Projekten ausgeht: man wird in einen Prozess kollektiven Lernens eintreten, den Paul Singer und Clarita Müller-Plantenberg erläutern und entwickeln in dem Konzept der Inkubation und Solidarischen Ökonomie, die Eigeninitiative, Selbstbestimmung und Selbstverwaltung von lokalen Gemeinschaften und die Öffnung für eine Neu-Konzeptualisierung und Institutionalisierung einschließt. So ein Prozess ist nur mit einem interdisziplinären Rahmen möglich. Dieser Rahmen soll auf einer zusätzlicher Kommunikation zwischen den Akteuren und Sozialwissenschaften beruhen und mit jenen, die auf dem Feld der Technologie arbeiten. So können dann technische „Experten“ angesichts eines technischen Projektfehlers, der aufgrund von bestimmten sozialen und kulturellen Realitäten zustande kam sagen, „es ist kein Scheitern ; es war ein Schritt innerhalb eines Lernprozesse der in die technischen Projekte und Studien einbezogen werden muss“. Wir haben das Energiethema gewählt; und insbesondere, Energie für eine nachhaltige ländliche Entwicklung in Subsahara-Afrika, um den Weg von der Theorie in die Praxis zu illustrieren und experimentell auszuprobieren, den Weg von den Beobachtungen zu der Veränderung, wobei Fragen, Annahmen, Strategien und konkrete Aktionen für den Wandel behandelt werden. Wir nennen unseren experimentellen Weg: DRIEE, das heißt auf Deutsch Ländliche Entwicklung und Inkubation von Energieunternehmen. Dabei gehen wir davon aus, dass: - Energie im Allgemeinen auf der internationalen Ebene fast gleichbedeutend mit Elektrizität ist. Heute bestehen die wichtigsten Bedürfnisse nach Energie dort wo die agro-pastorale Produktion, das Kochen, die Nahrungsmittelkonservierung und Verarbeitung …etc. stattfindet. - Diese ländliche Bevölkerung zu etwa 80% der nationalen Wirtschaft ausmacht. Dass sie gleichzeitig aber nur zu weniger als 5% der Energieproduktion Zugang hat, was oft auf Licht reduziert ist und nicht einmal ihrer Produktion zugute kommen kann. - Die Projekte für Energie und Elektrizität vor allem auf die Technologischen Fragen konzentriert sind und weniger auf die Bedürfnisse. Fast die Gesamtheit der Fonds für Energie wird in Bezug auf die Investitionen Infrastruktur der Produktion und Verteilung durch die konventionellen zentralisierten Netze geplant. Angesichts dieser Analysen gehen die in dieser Arbeit vorgenommenen Studien in Gambia und Kamerun von Bestandsaufnahmen und / oder beschreibenden regionalen Analysen aus: - von Bedürfnissen, von Praktiken und lokalen Initiativen von Fragen der Energie, für einzelne Professionen, Haushalte, Gruppen, spezifische Gruppen, wie Frauen, ländliche Gemeinden mit ihren spezifischen Charakteristika. - Von Potentialen: natürliche lokale Energieressourcen, soziokulturelle Ressourcen – so z.B. die empirisch feststellbaren menschliche Ressourcen wie endogenes Wissen und praktische organisatorische Fähigkeiten gegenüber den Problemen der Energie. Dieser experimentelle Schritt von Handlungsforschung (DRIEE) in Kamerun führte zu der Gründung einer Organisation, über die und mit der wir die Logik der Inkubation und Solidarischen Ökonomie einführen. Das ist FERDEDSI, das heißt auf Deutsch „Forum für Erneuerbare Energie – Nachhaltige Entwicklung und Internationale Solidarität“. Zunächst war dies eine Energiegenossenschaft und dann (im Prozess) wurde es zu einer institutionellen Nische von mehreren Mikro Initiativen in ländlichen Gebieten. FERDEDSI ist ein Prozess der Inkubation und ein Inkubator ist also gleichzeitig ein inkubiertes Energieunternehmen aber auch ein Inkubator für lokale Organisationen. Die ersten Aktionen finden in den Departments von Noun und Ménoua in der westlichen Provinz von Kamerun statt. Während der Forschungsperiode findet akademische Austausch statt (Nord-Süd und Süd-Süd), diese ist dabei zu formalen Partnerschaften zu werden, nicht nur zwischen Universitäten sondern genauer lokale Organisationen und Universitäten. Dieser letzte Typ von Partnerschaften, die die solidarische Ökonomie ausmachen ist auch eine Innovation des Prozesses für die afrikanischen Fälle, die dem Beispiel dessen, was in Lateinamerika geschieht, folgen. So kommt es zu gegenseitiger sinnvoller Ausbildung in den internationalen Arbeitsgruppen und Seminaren der Universität.

Diversity, local uses and availability of medicinal plants and wild yams in the Mahafaly region of south-western Madagascar

Short summary: This study was undertaken to assess the diversity of plant resources utilized by the local population in south-western Madagascar, the social, ecological and biophysical conditions that drive their uses and availability, and possible alternative strategies for their sustainable use in the region. The study region, ‘Mahafaly region’, located in south-western Madagascar, is one of the country’s most economically, educationally and climatically disadvantaged regions. With an arid steppe climate, the agricultural production is limited by low water availability and a low level of soil nutrients and soil organic carbon. The region comprises the recently extended Tsimanampetsotsa National Park, with numerous sacred and communities forests, which are threatened by slash and burn agriculture and overexploitation of forests resources. The present study analyzed the availability of wild yams and medicinal plants, and their importance for the livelihood of the local population in this region. An ethnobotanical survey was conducted recording the diversity, local knowledge and use of wild yams and medicinal plants utilized by the local communities in five villages in the Mahafaly region. 250 households were randomly selected followed by semi-structured interviews on the socio-economic characteristics of the households. Data allowed us to characterize sociocultural and socioeconomic factors that determine the local use of wild yams and medicinal plants, and to identify their role in the livelihoods of local people. Species-environment relationships and the current spatial distribution of the wild yams were investigated and predicted using ordination methods and a niche based habitat modelling approach. Species response curves along edaphic gradients allowed us to understand the species requirements on habitat conditions. We thus investigated various alternative methods to enhance the wild yam regeneration for their local conservation and their sustainable use in the Mahafaly region. Altogether, six species of wild yams and a total of 214 medicinal plants species from 68 families and 163 genera were identified in the study region. Results of the cluster and discriminant analysis indicated a clear pattern on resource, resulted in two groups of household and characterized by differences in livestock numbers, off-farm activities, agricultural land and harvests. A generalized linear model highlighted that economic factors significantly affect the collection intensity of wild yams, while the use of medicinal plants depends to a higher degree on socio-cultural factors. The gradient analysis on the distribution of the wild yam species revealed a clear pattern for species habitats. Species models based on NPMR (Nonparametric Multiplicative Regression analysis) indicated the importance of vegetation structure, human interventions, and soil characteristics to determine wild yam species distribution. The prediction of the current availability of wild yam resources showed that abundant wild yam resources are scarce and face high harvest intensity. Experiments on yams cultivation revealed that germination of seeds was enhanced by using pre-germination treatments before planting, vegetative regeneration performed better with the upper part of the tubers (corms) rather than the sets of tubers. In-situ regeneration was possible for the upper parts of the wild tubers but the success depended significantly on the type of soil. The use of manure (10-20 t ha¹) increased the yield of the D. alata and D. alatipes by 40%. We thus suggest the promotion of other cultivated varieties of D. alata found regions neighbouring as the Mahafaly Plateau.