La détection, la surveillance et la diffusion de la résistance des bactéries aux antibiotiques est un enjeu majeur au niveau mondial depuis la découverte et la diffusion de bactéries multi résistantes, en particulier la résistance aux carbapénèmes, spécifiquement chez les Entérobactéries et les bactéries du genre Pseudomonas et Acinetobacter. L’émergence et la dissémination des pathogènes Gram- résistants aux carbapénèmes est un contributeur significatif de la morbidité et la mortalité du patient. Malgré les efforts radicaux dans le contrôle de l’infection et les améliorations dans le diagnostique moléculaire, les bacilles Gram- résistants aux carbapénèmes demeurent une formidable menace vue que quelques agents antimicrobiens sont actifs et très peu devraient être disponibles dans le futur proche.L’origine et la source des gènes de résistance dans le monde sont mal connues et des travaux récents suggèrent que les animaux domestiques et sauvages, l’environnement mais également le tube digestif des mammifères et des humains pourraient représenter un réservoir et une source importante de gènes de résistance susceptibles d’être transmissibles à l’homme. C’est dans cette optique que ce projet de thèse s’articule avec comme objectifs : (i) la réalisation d’études épidémiologiques moléculaires d’isolats cliniques et animales résistants aux carbapénèmes isolés dans le basin Méditerranéen et la caractérisation des supports moléculaires de cette résistance ; (ii) la description de nouveaux mécanismes de résistance a l’imipenème ; et enfin (iii) le séquençage de génomes d’isolats cliniques résistants aux carbapénèmes et l’analyse de ces derniers. / The detection, monitoring and dissemination of bacterial resistance to antibiotics are a major issue worldwide since the discovery and spread of multi-resistant bacteria, in particular resistance to carbapenems, specifically among Enterobacteriaceae and bacteria of the genus Pseudomonas and Acinetobacter.The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant contributor to patient morbidity and mortality. Despite radical efforts in infection control and improvements in molecular diagnostics, carbapenem-resistant Gram-negative bacilli remain a formidable threat as few antimicrobial agents are reliably active and very little is expected to be available in the near future.The origin and source of resistance genes in the world are not well known and recent works suggest that domestic and wild animals, the environment (soil, water, rivers ..) but also the digestive tract of mammals and humans could represent a reservoir and an important source of resistance genes that may be transmissible to humans.It is in this context that this thesis project articulates with the following objectives: (i) The achievement of molecular epidemiological studies on carbapenem-resistant clinical and animal isolates collected from countries in the Mediterranean basin (Lebanon, Libya, France) and the characterization of the genetic determinants of this resistance; (ii) the description of new resistance mechanisms to imipenem; and finally (iii) The genome sequencing of clinical isolates resistant to carbapenems, the analysis of these genomes and the identification of mechanisms and genetic supports of the resistance to carbapenems and other antibiotics.

A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. As plantas apresentam sistema de defesa induzível, com a finalidade de economizar energia. Desse modo, a resistência induzida em condições naturais representará custo apenas na presença do patógeno. Porém, plantas que investem seus recursos para se defenderem na ausência de patógenos arcarão com custos que refletirão na produtividade, uma vez que as alterações metabólicas que levam a resistência apresentam custo adaptativo associado, o qual pode pesar mais do que o benefício. O efeito negativo na produtividade ocorre principalmente onde indutores químicos são utilizados repetidas vezes ou em doses mais elevadas. Assim, em alguns casos podemos estar caminhando sobre uma estreita linha entre custo e benefício, onde a cura pode ser tão ruim quanto a própria doença. Neste trabalho foram conduzidos experimentos objetivando verificar alterações bioquímicas e fisiológicas, correlacionando-as com parâmetros de produção do feijoeiro entre a indução mediada por acibenzolar-S-metil (ASM), indutor químico, e, a mediada por Bacillus cereus, indutor biológico, antes da chegada do patógeno. Para tanto, foram avaliados plantas de feijão, induzidas por esses dois indutores e desafiadas com Xanthomonas axonopodis pv. phaseoli, para constatar a ocorrência do fenômeno da indução de resistência. Na ausência do patógeno, foram avaliados os parâmetros fisiológicos respiração e fotossíntese, determinada a atividade de enzimas envolvidas no processo de defesa como peroxidase, quitinase, β-1,3-glucanase, fenilalanina amônia-liase e polifenoloxidase e a atividade de enzimas envolvidas no catabolismo como proteases, a síntese de compostos do metabolismo secundário como fenóis totais e lignina, a síntese de compostos do metabolismo primário como proteínas e açúcares redutores. Também se avaliou o crescimento das plantas, a produtividade e parâmetros de produção e alguns parâmetros de qualidade dos grãos. Observou-se a ocorrência da indução de resistência em função da aplicação dos dois indutores utilizados, porém para o indutor ASM a indução de resistência estava associada a aumentos na atividade de peroxidase, quitinase, β-1,3-glucanase e proteases, aumento da síntese de lignina e redução no teor de fenóis, aumentos no teor de proteínas solúveis e de açúcares redutores nas folhas, redução do crescimento e da produtividade, aumento do teor de proteína dos grãos e redução do teor de amido nestes. Já o B. cereus apenas ocasionou aumento na atividade de peroxidase de forma atenuada e tendeu a aumentar a atividade de proteases, e reduzir o teor de proteínas nas folhas sem interferir no crescimento ou na produtividade, mas reduziu o teor de proteína dos grãos, mas aumentou o teor de amido nestes. Portanto, o indutor B. cereus , aparentemente alterou muito pouco o metabolismo do feijoeiro, sem interferir na produtividade e melhorando a qualidade da produção, enquanto que o indutor abiótico ASM alterou muito mais seu metabolismo, gerando um custo metabólico e redirecionando os fotoassimilados para investir em defesas, a custo da redução da produtividade. / The induction of systemic resistance involves the activation of latent resistance mechanisms in plants against pathogens in response to the treatment with biotic or abiotic agents. The plants present latent defense system that can be activated with the goal of saving energy. Thus, the induced resistance under natural conditions will represent cost only in the pathogen presence. In this way, plants that invest their resources to defend themselves in the absence of the pathogen will pay off with costs that will reflect in productivity, since the metabolic changes that led to resistance have associated fitness cost which could outweigh the benefit. The negative effects on plant productivity usually occur when chemical inducers are used repeatedly or in higher doses, mainly in the absence of the pathogen. Thus, we can say that in some cases we can be walking on a fine line between cost and benefit, where the cure may be as bad as the disease itself. In this work, experiments were carry out to verify biochemical and physiologic alterations, correlating them with production parameters of bean plants treated with acibenzolar-S-methyl (ASM), chemical inducer, or Bacillus cereus , biological inducer, before the pathogen arrival. Initially, bean plants were evaluated for induced resistance against Xanthomonas axonopodis pv. phaseoli when treated with the two inducers. In the absence of the pathogen, it was evaluated the physiological parameters respiration and photosynthesis and the activity of enzymes involved in the defense as peroxidase, chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase and polifenoloxidase and activity of enzymes involved in the catabolism as proteases, and the synthesis of compounds of the secondary metabolism as phenols and lignin, and the synthesis of compounds of the primary metabolism as proteins and sugars. The growth of the plants was evaluated as well as their productivity and production parameters. Some quality parameters of the grains were also evaluated. The occurrence of the resistance induced in the bean plants against the pathogens was observed for the two inducers. However, for the ASM the resistance induced was associated to increases in peroxidase, chitinase and β-1,3-glucanase activities, increase in the protease activity, increase in lignin synthesis and reduction in the phenol content, increase in soluble proteins and sugar content in the leaves, reduction of the growth and productivity, increasing the protein and reducing the starch content of the grains. The B. cereus only increased peroxidase activity in a lower way and showed a tendency to increase protease activity, and to reduce the protein content in the leaves without interfering in the growth or in the productivity, but it reduced the protein content and it increased the starch content of the grains. Therefore, the biotic inducer, B. cereus altered a minimum the metabolism of the bean plant, without interfering in the productivity and improving the quality of the production, while the abiotic inducer ASM altered its metabolism, generating a metabolic cost and consuming the plant photosyntathes to invest in defenses, causing a reduction in the productivity.

A cana-de-açúcar é uma das principais culturas agrícolas plantadas no Brasil e apresenta significativa importância sócio-econômica e agroindustrial ao país. O cenário mundial encontrase bastante favorável no que concerne à comercialização de seus dois principais produtos derivados, o açúcar e o álcool, impulsionando o desenvolvimento do setor sucroalcooleiro nacional. Neste sentido, o melhoramento genético da cana-de-açúcar aparece como base fundamental para o desenvolvimento de novas variedades para a manutenção e incremento dos agronegócios da agroindústria sucroalcooleira. Técnicas de engenharia genética, como a transformação genética nuclear, estão trazendo excelentes resultados no melhoramento genético da cultura, permitindo diminuir o custo e o tempo de obtenção de novas variedades. Baseando-se na importância em se obter variedades tolerantes a diferentes estresses bióticos e abióticos que induzem perturbações metabólicas e ativam o processo de morte celular programada, o presente trabalho teve por objetivo transformar geneticamente a variedade de cana-de-açúcar RB835089 com o cDNA do gene AtBI-1 isolado de Arabidopsis thaliana, visando suprimir a indução do mecanismo de morte celular sob condição de estresse. Para isto, calos embriogênicos foram utilizados como explante alvo, empregando-se dois métodos de transformação, a cotransformação por biolística, e o mediado por Agrobacterium tumefaciens no qual foram testadas duas técnicas: (a) inoculação direta dos calos em suspensão bacteriana; e (b) agrobiolística que é o bombardeamento dos calos com partículas de tungstênio seguido da inoculação em suspensão bacteriana. A proteína AtBI-1 (Bax inhibitor-1) apresenta homólogos em outros organismos e está localizada na membrana do retículo endoplasmático. Ela apresenta funções citoprotetoras modulando o mecanismo de morte celular programada induzida por estresses bióticos e abióticos. Como resultados deste trabalho, diferentes taxas de eficiência da transformação genética foram obtidas pelo método mediado por A. tumefaciens nas duas técnicas testadas, sendo que suas taxas foram superiores às alcançadas pelo método de co-transformação por biolística. A expressão heteróloga do cDNA do gene AtBI-1 em cana-de-açúcar atenuou a indução das vias de morte celular em presença do antibiótico tunicamicina, indutor do estresse no retículo endoplasmático, sendo comprovado pela maior tolerância ao estresse das plantas transgênicas quando comparadas com as plantas não transformadas que foram afetas no crescimento do sistema radicular, conteúdo de clorofila total, apresentando sintomas típicos de morte celular programada como clorose foliar e morfologia irregular das raízes, com consequente morte do sistema radicular. / Sugarcane is one of the main crops planted in Brazil and presents significant socioeconomic and agribusiness importance to the country. The world scene is quite favorable as regards the marketing of its two main products, sugar and alcohol, driving the development of the national sugar-alcohol sector. Therefore, the sugarcane genetic breeding appears as the fundamental base for developing new varieties for the maintenance and increase of agribusiness in the sugarcane agroindustry. Genetic engineering techniques, such as the nuclear genetic transformation, are providing excellent results in genetic breeding of this crop allowing reducing the cost and time to obtain new varieties. Based on the importance of obtaining varieties tolerant to different biotic and abiotic stresses that induce metabolic disturbances and activate the process of programmed cell death, this work aimed to transform sugarcane variety RB835089 with the cDNA of AtBI-1 gene, isolated from Arabidopsis thaliana, to suppress the induction of the cell death mechanism under stress condition. For this, embryogenic calli were used as target explant, by using two methods of transformation, the cotransformation by biolistic, and mediated by Agrobacterium tumefaciens in which two techniques were tested: (a) direct inoculation of calli in bacterial suspension; (b) agrobiolistic which is the bombardment of calli with tungstein particles followed by inoculation in bacterial suspension. The AtBI-1 protein (Bax inhibitor-1) presents homologs in other organisms and is located in the endoplasmic reticulum membranes. It has cytoprotective functions by modulating the mechanism of programmed cell death induced by biotic and abiotic stresses. As results of this work, different efficiency rates in genetic transformation were obtained in the method mediated by A. tumefaciens in the two techniques tested, and that their rates were higher than those achieved using the cotransformation by biolistic. The heterologous expression of cDNA of AtBI-1 gene in sugarcane attenuated the induction of cell death pathways in the presence of tunicamycin antibiotic, an inducer of stress in the endoplasmic reticulum, being proven by the increased stress tolerance of transgenic plants compared with sugarcane wild type that were affected in the root growth, total chlorophyll content, showing typical symptoms of programmed cell death such as leaf chlorosis and irregular morphology of the roots, with subsequent death of the root system.

An important challenge in natural language surface realization is the generation of grammatical sentences from incomplete sentence plans. Realization can be broken into a two-stage process consisting of an over-generating rule-based module followed by a ranker that outputs the most probable candidate sentence based on a statistical language model. Thus far, an n-gram language model has been evaluated in this context. More sophisticated syntactic knowledge is expected to improve such a ranker. In this thesis, a new language model based on featurized functional dependency syntax was developed and evaluated. Generation accuracies and cross-entropy for the new language model did not beat the comparison bigram language model.

Tesis para optar al grado de Mag?ster en Ling??stica menci?n Lengua Espa?ola / La tesis que a continuaci?n expongo ofrece un establecimiento cr?tico de la lectura de la Cr?nica de Jer?nimo de Vivar, ling??sticamente anotada y prologada. En ella doy cuenta de una visi?n descriptiva y anal?tica, no exhaustiva, de sus principales rasgos fon?ticos, morfol?gicos y sint?cticos. El estudio filol?gico-ling??stico se realiza sobre la base de una completa transcripci?n paleogr?fica de la obra, con el prop?sito de obtener un documento lo m?s fiel al original. Para ello, se contrastaron dos transcripciones de la misma: una de Leopoldo S?ez-Godoy (Coloquium Verlag, Berl?n, 1979) y otra realizada por el profesor Manuel Contreras Seitz. El estudio filol?gico, que se efectu? con la Cr?nica de Vivar, tuvo como modelos las ediciones cr?ticas realizadas por los profesores Ferreccio y Kordic en sus descripciones ling??sticas, dejando de lado la moderaci?n fonografem?tica y los aspectos literarios y referenciales por lo extenso que resultar?a un trabajo de este tipo. Para el estudio netamente ling??stico, realic? la revisi?n de la Historia del Espa?ol de Am?rica de Juan Antonio Frago, la Historia de la Lengua Espa?ola de Rafael Lapesa y la Gram?tica Hist?rica Espa?ola de Vicente Garc?a de Diego, con el fin de identificar los principales rasgos idiom?ticos meridionales presentes en la obra de Vivar; y, establec?, por contraste, los rasgos caracter?sticos del centro norte espa?ol (Burgos, lugar hipot?tico de nacimiento del autor) presentes en la Cr?nica, tomando como base fuentes sobre el espa?ol peninsular como las obras ya citadas de Lapesa y Garc?a de Diego.

La première partie de ma thèse est la description d'un modèle algébrique pour la gestion des séquences temporelles synchronisées. Ce modèle est une extension du modèle relationnel classique auquel on ajoute un type nouveau, le type séquence temporelle. L'algèbre relationnelle est augmentée de trois opérateurs dédiés à ce nouveau type. Ces opérateurs permettent de retrouver toutes les opérations classiquement conduites sur des séquences temporelles. Le langage utilisateur correspondant est exposé, ainsi que de nombreux exemples, puisés notamment dans le domaine de la gestion des partitions symboliques. La seconde partie est la description d'un index permettant de réaliser plusieurs types de recherches dans des partitions symboliques (exacte, transposée, avec ou sans rythme et approchée). Il repose notamment sur la notion de signature algébrique. Dans la dernière partie, je décris une plateforme dédiée à la gestion du contenu musical symbolique qui est une application des deux précédentes parties.

Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many pathogenic bacteria require heme and obtain it from their environment. Heme transverses the cytoplasmic membrane via an ATP binding cassette (ABC) pathway. Although a number of heme ABC transport systems have been described in pathogenic bacteria, there is as yet little biophysical characterization of the proteins in these systems. The sia (hts) gene cluster encodes a heme ABC transporter in the Gram positive Streptococcus pyogenes. The heme binding protein (HBP) of this transporter is SiaA (HtsA). Several biophysical techniques were used to determine the coordination state, and spin state of both the ferric and ferrous forms of this protein. Identifiers from these techniques suggested that the heme is six-coordinate and low spin in both oxidation states of the protein, with methionine and histidine as axial ligands. The pKa of SiaA was determined, as were the reductive and oxidative midpoint potentials. Guanidinium titration studies of wild-type SiaA showed that the ferric state is less stable than the ferrous state. Free energy of unfolding values [ÄG(H2O)] for the oxidized and reduced proteins were 7.3 ± 0.8 and 16.0 ± 3.6 kcal mol−1, respectively. Denaturation of the histidine mutant H229A was not able to be followed via absorbance spectrometry, possibly due to the large amount of apoprotein present or to non-specific binding of the heme in the binding pocket. The biophysical characterization described herein will significantly advance our understanding of structure-function relationships in HBP.

Bakgrund: Tidigare studier visar att faktorer som exempelvis rökning och låg utbildningsnivå har samband med ett lägre frukt- och grönsaksintag. För att öka frukt- och grönsakskonsumtionen är det värdefullt att identifiera grupper med lågt intag. Syfte: Syftet med studien var att undersöka hur frukt- och grönsaksintaget såg ut hos vuxna samt om livsstilsfaktorer och sociala faktorer kan ha samband med frukt- och grönsaksintaget. Metod: En enkätstudie genomfördes i Umeå och Örnsköldsvik där 275 vuxna i ett bekvämlighetsurval tillfrågades att delta. Efter externt bortfall och efter exklusion av ofullständigt ifyllda enkäter ingick totalt 101 deltagare i studien. Enkäten bestod av 20 frågor, varav 19 slutna. Data bearbetades och analyserades i SPSS 17.0 med chi-2 test, signifikansnivån sattes till p<0,05. Resultat: Majoriteten av respondenterna hade ett rapporterat intag lägre än rekommendationen (74 %). Av respondenterna hade 40 individer ett lågt frukt- och grönsaksintag (<3ggr/dag), 19 individer hade ett medelhögt intag (3ggr/dag) och 42 individer hade ett högt intag (>3ggr/dag). En signifikant större andel av respondenterna med högt frukt- och grönsaksintag fanns i grupperna med hög utbildningsnivå, hög fysisk aktivitet, lågt alkoholintag, icke rökare, kunskap om Livsmedelsverkets kostråd om 500 gram frukt och grönsaker per dag samt de som ansåg att detta kostråd var viktigt. Slutsats: Resultaten från denna studie tyder på att ytterligare insatser för att främja ett högre frukt- och grönsaksintag behövs, framförallt i de grupper som tenderar att ha lägst intag. Livsstilen kan ha större påverkan på frukt- och grönsaksintaget än enskilda faktorer. Sambandet mellan utbildningsnivå och frukt- och grönsaksintag gör att det blir viktigt att identifiera vilka yrkesgrupper som representerar lägre utbildningsnivåer för att se var fler insatser behövs. / Background: Earlier studies have shown associations between factors such as smoking and low education level and a lower fruit and vegetable intake. To increase fruit and vegetable consumption it is important to identify groups with low intake. Objective: The aim of this study was to investigate the intake of fruit and vegetables among adults and if lifestyle and social factors may be associated with this intake. Method: A study was conducted in Umeå and Örnsköldsvik where convenience samples of 275 adults were asked to participate. After external loss and after exclusion of incomplete questionnaires there were a total of 101 participants in the study. The survey was based on 20 questions, of which 19 were closed. The data was processed and analyzed in SPSS 17.0 with chi-square tests, the level of significance was set to p<0,05. Results: The majority of respondents reported an intake below the recommendation (74%). Of all respondents, 40 participants had low fruit and vegetable intake (<3times/day), 19 had moderate intake (3 times/day) and 42 respondents had high intake (>3 times/day). A significant higher proportion of respondents with high fruit and vegetable intake where found in the groups with high level of education, high physical activity, low alcohol intake, non- smoking, awareness of the National Food Agency´s dietary advice about 500 gram fruit and vegetables per day and the opinion that this advice was important. Conclusion: The results from this survey suggest that further efforts to promote a higher fruit and vegetable intake are needed, especially in the groups who tend to have the lowest intake. Lifestyle may have a greater impact on fruit and vegetable intake than single factors alone. The association between level of education and fruit and vegetable intake makes it important to identify professions who represent a lower education level to see where more effort is needed.

Endotoxin or Lipopolysaccharide (LPS) is the invariant structural component of gram negative bacterial outer membranes and is the chief causative factor of Sepsis or endotoxic shock. Sepsis is a syndrome that has very high mortality rates even in this age of excellent therapeutics and critical patient care. The treatment for sepsis till date remains nonspecific and supportive due to lack of effective anti-endotoxic drugs. Sepsis is initiated when the circulating bacteria shed LPS from their cell envelopes. Shed LPS aggregates are recognized by LPS binding proteins and receptors, which activate the host's immune system. Uncontrolled and excessive stimulation of the host's immune system precipitates endotoxic shock which in advanced cases involving multiple system organ failure inevitably lead to patient's death. Many strategies have been tested out to combat this deadly affliction. One of the attractive clinical modalities in sepsis treatment is the use of peptides as LPS sequestering anti-endotoxic drugs. A classical peptide antibiotic of this class is Polymyxin B (PMB) a cyclic cationic acylated molecule, that recognizes LPS with a very high affinity. This thesis describes kinetics and thermodynamics of PMB-LPS interactions and applies these parameters over a framework of different models so as to gain insights into the structure-function relationships that govern the interactions of this peptide with endotoxin(s). Classical biophysical techniques like fluorescence, circular dichroism spectroscopy, stopped flow kinetics, titration calorirnetry (ITC) and the relatively new technique of Surface Plasmon Resonance (SPR) have been employed to dissect out the mechanism of the range of non-covalent forces that are involved in peptide-endotoxin recognition. Certain proteins that exhibit LPS binding activity have also been studied to gains insight about their mode of action. Implications of these studies for designing peptides that have better anti-endotoxic properties are also highlighted. The first chapter introduces and highlights the clinical features of sepsis. It also attempts to shed light on the LPS mediated signal transduction pathway that leads to endotoxic shock. This chapter also briefly explains the roles of many LPS receptors that are present in the human system and their specific roles in the signal transduction pathways. The second part of this chapter deals with the role of cationic peptides as anti-endotoxic drugs. Certain key functional aspects of these peptides, which impart in them, the desirable property of LPS recognition have also been discussed The second chapter describes the kinetic studies undertaken to unravel the exact mechanism of LPS-PMB interaction. The studies reveal that PMB recognizes LPS in a biphasic manner, with the second, unimolecular isomerization step of the reaction being the rate-limiting step. The initial reaction is shown to be influenced by the presence of salt in the reaction medium. The dissociation phase of this interaction also shows a biphasic pattern. These data allow us to speculate upon the exact mechanism by which PMB is able to recognize LPS. The studies also shed light on some structural aspects that govern and confer such high LPS binding activity to PMB. Based on these a model has been proposed to explain this recognition (C.J. Thomas et al, 1998). The second chapter discuses the mode of action of various PMB analogs. These analogs have been chosen in terms of their mode of action as well as their structural similarly to PMB. The affinities of these analogs to LPS and lipid A were quantified using the Surface plasmon resonance (SPR) method. SPR, a technique that relies on the quantification of change in mass during a binary binding process occurring between an immobilized entity and a flowing ligand, is a rapid and sensitive method to measure biologically relevant interactions. SPR studies provide us with the binding constants and thermodynamic parameters that allow evaluation of the affinities of these peptides towards LPS (C.J.Thomas and A.Surolia, 1999). The third chapter discusses a hitherto unknown mode by which PMB acts on a LPS lamellae. The results of this study wherein the binding affinities of PMB and its analogs were performed on monolayers and tethered liposomes, show that PMB is able to remove specifically LPS or lipid A from monolayers or bilayer assemblies such as tethered liposomes. The exact mode of action of PMB is deciphered in the light of these new studies, which allow us to posit on the observed efficacy of PMB in neutralizing the endotoxin as compared to peptides with nearly similar affinities for LPS (C.J Thomas et al 1999). In the fourth chapter a series of 23 residue peptides, based on the sequence corresponding to the anti-sense strand of magainin gene have been synthesized. Magainin an amphiphilic helical peptide obtained from frog skins plays a vital role in the innate immune defense mechanisms of these organisms. It also exhibits LPS binding activity that makes it an attractive target as an anti-endotoxic drug. Biochemical and biophysical characterization of these peptides reveal that they have the tendency to perturb both the inner and the outer membranes of E.coli. The peptides are amphiphilic and have helical structure in a membrane bound environment. Three of the peptides tested have high affinities for lipid A that approach the values shown by PMB. The kinetic parameters obtained by stopped flow and SPR studies in conjunction with the therrnodynamic parameters obtained using ITC studies allow us to highlight the key structural features that need to be exhibited by peptides that are designed to be LPS recognizers. The studies also project the fact that ionic forces play an important role in the initial recognition of LPS by these peptides. Fortification of the might of these ionic charges increases affinity for LPS where as the hydrophobic residues that interact at the next phase of binding are more amenable to disruptions in contiguity. These factors are discussed using the helical wheel diagram that shows the clear amphiphilicity displayed by these peptides. (C.J Thomas et al Manuscript under preparation, 2000) Chapter six discusses the mode of action of certain LPS binding proteins. Limulus anti endotoxic factor (LALF) plays a vital role in the innate immune based defense systems of the horseshoe crab. Galectin-3 is a metal ion independent, galactosc binding Icctin of human origin with unknown functions. Both these phylogcntically-unrclatcd proteins exhibit LPS/lipid A recognizing properties. ITC and SPR studies have been used to determine the binding constants displayed by these proteins for lipid A. LALF bind to lipid A with very high affinity than compared to Galectin-3 and is also able to take away selectively lipid A from both monolayers and tethered liposomes. Galectin-3 does not show this property of LALF, which might account for its lowered affinities. Also structurally LALF has amphiphilic nature that confers high lipid A binding activity, which is clearly lacking in Galectin-3. These studies in conjunction with the knowledge gained from the study of LPS-PMB interaction stress on the importance of amphiphilicity in LPS recognition. (C.J Thomas et al Manuscript under preparation, 2000). The final chapter is a general discussion that attempts to collate all these kinetic and thermodynamic observations in the pursuit of designing small easily manipulatable peptides that exhibit high LPS binding activity. These studies are aimed to act as rough guidelines to the design of LPS sequestering peptides that might have better therapeutic and pharmacokinetic properties. The appendix to the main body of work presented in thesis are two pieces of work pertaining to the elucidation the kinetics and mechanism of sugar lectin interactions, when sugars are presented as glycolipids in monolayers or bilaycrs liposomes. Mode of the presentation of sugars at cell-surfaces in the form of glycolipids as ligands influence their recognition by macromolecular receptors like lectins. Appendix 1 is a study of the mode of action of Ulex europeus I lectin binding to H-fucolipid containing tethered liposomes, by SPR. Fucosylated sugars are often used as key markers in histochemical analysis of malignant cancerous tissues. Ulex lectin plays a vital role as a marker for identification of these tissues. The kinetics and thermodynamic parameters that are obtained in this study throw some light on the mode of recognition of glycolipid receptor by Ulex europeus I lectin (C.J Thomas and A. Surolia 2000). Appendix 2 is a study, that attempts to quantify the initial kinetic parameters that correlate the recognition of glycolipid receptors with their inclination at the membrane surface and the influence of charge on them by soyabean agglutinin (SBA), Abrus agglutinin I and II. Studies on the soyabean agglutinin-globoside interaction highlights the divalent cation mediated reorientation of these receptors on their accessibility and recognition to the agglutinin. The divalent cations are speculated to orient the oligosaccharide head groups in a spatial geometry that allows a heightened kinetics of their interaction by SBA. These studies reveal that the reorganization of the binding pocket of a lectin can also have a profound influence on ihc rates of recognition of a glycospingolipid ligand by a lectin as exemplified by Abrus agglutinin II- GM1 interactions (C.J Thomas ct al, Manuscript under preparation).

Au cours de la dernière décennie, d'énormes quantités de documents multimédias ont été générées. Il est donc important de trouver un moyen de gérer ces données, notamment d'un point de vue sémantique, ce qui nécessite une connaissance fine de leur contenu. Il existe deux familles d'approches pour ce faire, soit par l'extraction d'informations à partir du document (par ex., audio, image), soit en utilisant des données textuelles extraites du document ou de sources externes (par ex., Web). Notre travail se place dans cette seconde famille d'approches ; les informations extraites des textes peuvent ensuite être utilisées pour annoter les documents multimédias et faciliter leur gestion. L'objectif de cette thèse est donc de développer de tels modèles d'extraction d'informations. Mais les textes extraits des documents multimédias étant en général petits et bruités, ce travail veille aussi à leur nécessaire robustesse. Nous avons donc privilégié des techniques simples nécessitant peu de connaissances externes comme garantie de robustesse, en nous inspirant des travaux en recherche d'information et en analyse statistique des textes. Nous nous sommes notamment concentré sur trois tâches : l'extraction supervisée de relations entre entités, la découverte de relations, et la découverte de classes d'entités. Pour l'extraction de relations, nous proposons une approche supervisée basée sur les modèles de langues et l'algorithme d'apprentissage des k-plus-proches voisins. Les résultats expérimentaux montrent l'efficacité et la robustesse de nos modèles, dépassant les systèmes état-de-l'art tout en utilisant des informations linguistiques plus simples à obtenir. Dans la seconde tâche, nous passons à un modèle non supervisé pour découvrir les relations au lieu d'en extraire des prédéfinies. Nous modélisons ce problème comme une tâche de clustering avec une fonction de similarité là encore basée sur les modèles de langues. Les performances, évaluées sur un corpus de vidéos de matchs de football, montrnt l'intérêt de notre approche par rapport aux modèles classiques. Enfin, dans la dernière tâche, nous nous intéressons non plus aux relations mais aux entités, source d'informations essentielles dans les documents. Nous proposons une technique de clustering d'entités afin de faire émerger, sans a priori, des classes sémantiques parmi celles-ci, en adoptant une représentation nouvelle des données permettant de mieux tenir compte des chaque occurrence des entités. En guise de conclusion, nous avons montré expérimentalement que des techniques simples, exigeant peu de connaissances a priori, et utilisant des informations linguistique facilement accessibles peuvent être suffisantes pour extraire efficacement des informations précises à partir du texte. Dans notre cas, ces bons résultats sont obtenus en choisissant une représentation adaptée pour les données, basée sur une analyse statistique ou des modèles de recherche d'information. Le chemin est encore long avant d'être en mesure de traiter directement des documents multimédia, mais nous espérons que nos propositions pourront servir de tremplin pour les recherches futures dans ce domaine.