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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1984 C68 / Master of Science

Cette étude s’inscrit dans le cadre de l’amélioration des connaissances sur la dégradation photocatalytique des bioaérosols bactériens. La photocatalyse est une technique d’épuration basée sur l’excitation d’un semi-conducteur par un rayonnement le plus généralement ultraviolet. Cette technologie permet, en théorie, de minéraliser pas à pas les polluants. Or, si les conditions optimales ne sont pas réunies, la minéralisation incomplète peut conduire à des sous-produits de dégradation de toxicité potentiellement préoccupante.L’objectif de ces travaux a donc été d’apporter des éléments de compréhension quant aux mécanismes de dégradation photocatalytique d’un bioaérosol bactérien modèle d’E.coli, où de nombreux phénomènes sont couplés. Ainsi, pour distinguer les différents processus mis en jeu, deux approches expérimentales ont été menées. La première, nommée approche « batch », a permis d’isoler la réaction photocatalytique, à proprement parler, en étudiant les étapes d’inactivation, de libération de sous-produits et de minéralisation progressive. La seconde, appelée approche « dynamique » a permis quant à elle la mise en place d’un dispositif expérimental adapté à la dégradation photocatalytique d’un bioaérosol d’E.coli. Les capacités de la photocatalyse à inactiver et minéraliser des espèces bactériennes ont pu être démontrées. Les paramètres clés d’une dégradation efficace ont été mis en évidence et ont permis de décrire les verrous indispensables à une industrialisation sûre du procédé / This study comes within the scope of improving knowledge concerning the photocatalytic degradation of bacterial bioaerosol. Photocatalysis is a purification technology generally based on the excitation of a semiconductor by an ultraviolet radiation. This technology can, in theoretical ways, mineralize pollutants step by step. However, if optimal conditions are not gathered, this mineralization is incomplete and can lead to the formation of potentially toxic by-products. The aim of this work was therefore a better understanding of the mechanisms of photocatalytic degradation of a bacterial bioaerosol of E.coli, where numerous phenomenon are linked. Thus, to distinguish the different processes, two experimental approaches were used. The first one, called “batch approach”, allowed to consider the photocatalytic reaction itself, by studying the steps of inactivation, by-products formation and progressive mineralization. The second one, named “dynamic approach”, consisted to design an experimental setup suited to the photocatalytic degradation of a bioaerosol of E.coli. The abilities of photocatalysis to inactivate and mineralize bacteria could be demonstrated. The key parameters of an efficient degradation were highlighted and allowed to underline the problems to solve before having a safe industrialization of the photocatalysis

Ph.D. / Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).

O objetivo do presente trabalho foi quantificar, in vitro e in vivo, a endotoxina bacteriana (LPS) aderida a ligaduras ortodônticas elastoméricas estéticas de poliuretano e de silicone, empregando o teste Limulus Amebocyte Lysate (LAL). Para o estudo in vitro foram utilizadas quatro tipos de ligaduras elastoméricas estéticas: Sani-Ties (poliuretano) e Sili-Ties (silicone), da GAC, e Mini Single Case Ligature Stick (poliuretano) e Synergy&reg; Low-friction ligatures (silicone), da Rock Mountain, sendo 5 contaminadas com solução de endotoxina (controle positivo) e 5 não-contaminadas (controle negativo). Réplicas feitas de fio de amarrilho torcido e de aço inox fundido, de mesmo tamanho e formato das ligaduras elastoméricas, contaminadas ou não com endotoxina, foram usadas como controle. A quantificação de endotoxina foi realizada por meio do teste LAL (Kit QCL-1000&trade;), sendo os resultados expressos em unidades de endotoxina (UE/mL). No estudo in vivo, 20 pacientes de ambos os gêneros, com faixa etária entre 15 e 30 anos, que iniciaram tratamento com aparelho ortodôntico fixo na Faculdade de Odontologia de Ribeirão Preto - Universidade de São Paulo receberam, randomicamente, em quadrantes alternados, os mesmos quatro tipos de ligaduras elastoméricas utilizadas no estudo in vitro, sendo uma ligadura de cada marca inserida nos caninos superiores e inferiores (13, 23, 33 e 43), aleatoriamente. Vinte e um dias após, as ligaduras foram removidas e processadas para quantificação da endotoxina bacteriana, utilizando também o Kit QCL-1000&trade;. Todos os dados obtidos foram submetidos à análise estatística apropriada de acordo com a distribuição dos dados, por meio dos testes de Kruskal-Wallis e pós-teste de Dunn (estudo in vitro) e ANOVA de medidas repetidas e pós-teste de Tukey (estudo in vivo). Todas as análises foram efetuadas por meio do programa Graph Pad Prism 4.0, com nível de significância de 5%. De acordo com os resultados obtidos, observou-se que a endotoxina bacteriana aderiu-se a todos os materiais testados. No estudo in vitro, o grupo GAC silicone foi o que apresentou menor mediana de contaminação (1,15 UE/mL), com relação aos outros grupos (p<0,0001), os quais não apresentaram diferença estatisticamente significante quando comparados entre si (p>0,05). No estudo in vivo, de maneira semelhante ao observado no estudo in vitro, o grupo GAC silicone foi o que apresentou menor média de contaminação (0,577±0,017 UE/mL), com diferença estatisticamente significante (p<0,001) em comparação aos demais grupos. Pôde-se concluir, que a endotoxina bacteriana apresentou afinidade pelas ligaduras elastoméricas estéticas à base de silicone e poliuretano testadas. As ligaduras de silicone da marca GAC foram as que apresentaram menor quantidade de endotoxina aderida às suas superfícies / The objective of this study was to quantify, in vitro and in vivo, bacterial endotoxin (LPS) attached to esthetic elastomeric orthodontic ligatures made of polyurethane and silicone using the Limulus Amebocyte Lysate test (LAL). For the in vitro study, were used four types of aesthetic elastomeric ligatures: Sani-Ties (polyurethane) and Sili-Ties (silicone) - GAC, and Mini Single Case Ligature Stick (polyurethane) and Synergy&reg; Low-friction ligatures (silicone) - Rock Mountain; 5 of each were contaminated with endotoxin solution (positive control) and 5 non-contaminated (negative control). Replicas made of twisted wire ligatures and of cast stainless steel, of the same size and shape than the elastomeric ligatures, contaminated or not with endotoxin, were used as control. Endotoxin quantification was performed using the LAL test (QCL-1000&trade; kit), the results being expressed in EU/mL. In the in vivo study, 20 patients of both genders, with ages ranging from 15 to 30 years old, who started treatment with a fixed orthodontic appliance at the School of Dentistry of Ribeirão Preto - University of São Paulo, received randomly the same four types of elastomeric ligatures used in the in vitro study, being a ligature of each brand inserted in the upper and lower canines (UR3, UL3, LR3, LL3), randomly. Twenty-one days later, ligatures were removed and processed for quantification of bacterial endotoxin, using the same test as the in vitro study. All data were submitted to appropriate statistical analysis according to the data distribution, using KruskalWallis tests and Dunn post-test (in vitro study) and ANOVA of repeated measurements and Tukey\'s post-test (in vivo study). All analyzes were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. According to the results obtained, it was observed that the bacterial endotoxin attached to all materials tested. In the in vitro study, the GAC silicone group had the lowest median contamination (1.15 EU/mL), in relation to the other groups (p<0.0001), which did not present a statistically significant difference when compared to each other (p>0.05). In the in vivo study, similar to that observed in the in vitro study, the GAC silicone group had the lowest mean contamination (0.577 ± 0.017 EU/mL), with a statistically significant difference (p< 0.001) compared to the others groups. It could be concluded that bacterial endotoxin exhibited affinity for the tested silicone and polyurethane aesthetic elastomeric ligatures. The silicone ligatures of GAC brand were the ones that presented less amount of endotoxin attached to their surfaces

INTRODUÇÃO: A poluição do ar traz diversos efeitos à saúde e, em particular, o material particulado menor do que 2,5 &#956;m (PM2,5), está associado ao aumento das taxas de morbidade e mortalidade devido a doenças cardiorespiratórias. A maioria dos estudos foca apenas na composição química do material, porém os componentes do bioaerossol podem ser responsáveis por aproximadamente 22% da massa total. OBJETIVOS: Nesse estudo, objetivamos determinar a contribuição relativa de fungos e endotoxinas (constituinte da parede celular de bactérias gram-negativas) no PM2,5 e verificar possíveis efeitos inflamatórios locais devido à exposição a fungos e PM2,5 em ratos. MÉTODOS: O estudo foi dividido em três partes: 1) comparação de metodologias de coleta de fungos em amostradores de ar de curta e longa duração realizada em ambiente externo e em ambiente de concentrador de partículas ambientais em Boston, MA. 2) Coleta de 4 tipos de filtros a partir de amostrador de ar de PM2,5 utilizados para análise separadamente de fungos, quantificação de endotoxinas, análise de elementos químicos e extração para instilação em ratos em São Paulo. Dados meteorológicos também foram coletados. 3) Instilação intratraqueal de PM2,5 e fungos originários da atmosfera de São Paulo em ratos machos Wistar divididos em 3 grupos: A (administração de 6,3x102 esporos/&#956;g de PM2.5), B (18x102 esporos/&#956;g de PM2.5) e C (controle - instilação a partir de extração de filtro branco). O sacrifício foi feito após 24 horas da exposição, depois da retirada do lavado broncoalveolar para contagem total e diferencial de leucócitos, além de quantificação de citocinas de respostas TH1 e TH2. RESULTADOS: O estudo de comparação de metodologias mostrou que, entre os amostradores de ar de curta duração, Personal Burkard recuperou maior diversidade e concentração do que Andersen (p<0,05). Entre os amostradores de longa duração, Recording Burkard mostrou-se o melhor em termos de diversidade e concentração de esporos do que filtros MCE (p<0,05). Também observamos que os fungos representaram relevante porção do PM2,5, chegando a concentrações de 2159 esporos/&#956;g em ambiente de Concentrador de Partículas alocado em Boston, MA. A segunda parte do estudo mostrou novamente que os fungos representam porção relevante do PM2.5 alcançando valores médios de até 1345 esporos/&#956;g de PM2,5 a partir de coletas da atmosfera de São Paulo. Da mesma forma, endotoxinas foram obtidas em concentrações médias de 5,52 EU/&#956;g de PM2,5. Os modelos de regressão linear múltipla mostraram que a contagem total de fungos, de basidiósporos hialinos e de Cladosporium sp foi correlacionada positivamente com a presença do fator Ba/Ca/Fe/Zn/K/Si no PM2,5 (p<0,05). Os gêneros Penicillium/Aspergillus foram correlacionados positivamente à concentração de material particulado na atmosfera (p<0,05). Os ascósporos sem pigmentação foram correlacionados positivamente à umidade (p<0,05). As endotoxinas foram correlacionadas apenas à temperatura atmosférica (p<0,05). O modelo de instilação em animais mostrou que a administração de 6,3x102 esporos/&#956;g de PM2,5 foi responsável por um aumento na concentração de TNF e IFN no lavado broncoalveolar (p<0,05). Não foi observado o mesmo efeito quando a concentração de esporos foi de 18x102 esporos/&#956;g de PM2,5. Não foram observadas diferenças sgnificativas contagem total e diferencial de leucócitos. CONCLUSÕES: Fungos e endotoxinas são responsáveis por fração relevante do PM2,5. Esses contaminantes biológicos podem estar associados não apenas a fatores meteorológicos, mas também a elementos químicos presentes no Material Particulado, especialmente a elementos sinalizadores de tráfego de veículos e ressuspensão da crosta. Exposições a diferentes concentrações de fungos associados ao PM2,5 em ratos não sensibilizados podem levar a diferenças em respostas inflamatórias agudas, porém os mecanismos responsáveis por tais resultados devem ser objeto de estudos futuros. / INTRODUCTION: It is well known that air pollution is associated with health effects, particularly, particulate matter (PM) is correlated with increment in morbity and mortality by cardiorespiratory diseases. A lot of studies have focused on chemical characterization of PM, although components of bioaerosols may comprise nearly 22% of the total mass. OBJECTIVES: The aims of this study were to determine the relative contribution of fungi and endotoxin (component of cell wall in gram-negative bacteria) in PM2.5 and verify acute local inflammatory response due to fungi and PM2.5 exposure in rats. METHODS: The study was divided in 3 parts: 1) Comparison among short and long term samplers located outdoor and in Ambient Particle Concentrator located in Boston, MA. 2) Sampling of 4 different filters types analyzed for fungi, endotoxin, chemical composition and extraction for instillation experiments in Sao Paulo. Meteorological data were also collected. 3) Intratracheal instillation of PM2.5 and fungi in male Wistar rats, divided in 3 groups: A (administration of 6.3x102 spores/&#956;g of PM2.5), B (administration of 18x102 spores/&#956;g of PM2.5) and C (control - blank filter). The animals were sacrificed 24 hours after exposure to collect bronchoalveolar lavage. Total and differential counts of leukocytes as TH1 and TH2 cytokines quantification were assessed. RESULTS: Sampling techniques comparisons showed that, the short term sampler Personal Burkard was able to collect more diversity and concentration of spores than Andersen sampler (p<0.05). The long term sampler Recording Burkard was also able to collect more diversity and concentration of spores than MCE filters (p<0.05). We also observed that fungi contribution to PM2.5 was relevant, representing up to 2159 spores/&#956;g of PM2.5 in Ambient Particle Concentrator located in Boston, MA. The second part of the study demonstrated again that fungi represent a relevant portion of PM2.5, reaching average values of 1,345 spores/&#956;g of PM2.5 in Sao Paulo atmosphere. Also, endotoxin concentrations reached averages values of 5.52 EU/&#956;g of PM2.5. Multiple linear regression models showed that total fungi counts, hyaline basidiospores and Cladosporium sp were correlated with factor Ba/Ca/Fe/Zn/K/Si (p<0.05). The genera Penicillium/Aspergillus were correlated with PM2.5 mass (p<0.05). Colorless ascospores were correlated with relative humidity (p<0.05). Endotoxin was correlated only with temperature (p<0.05). Instilattion results showed that administration of 6.3x102 spores/&#956;g of PM2.5 was responsible for an increase in TNF and IFN in bronchoalveolar lavage (p<0.05). The same pattern wasn\'t observed when 18x102 spores/&#956;g of PM2.5 was administered. No significant differences were observed leucocytes total and differential counts. CONCLUSIONS: Fungi and endotoxin represent a relevant fraction of PM2.5. These biological contaminants can be associated not only with meteorological parameters, but with elementary composition, especially elements of traffic and crustal ressuspension. Acute exposure of non sensitized rats to different concentrations of fungi associated to PM2.5 might trigger different inflammatory responses, although the specific mechanisms remain unknown and further research is needed.

Mesmo com o constante avanço no estudo da sibilância e asma, existem inúmeras controvérsias sobre a participação da exposição à endotoxina, background genético e ativação celular. Investigamos a participação da exposição à endotoxina ambiental e o papel do LPS no desenvolvimento dos fenótipos de sibilância e asma. Para tanto selecionamos crianças sibilantes e não sibilantes, e crianças asmáticas e não asmáticas, sendo seu sangue coletado e as PBMC cultivadas com LPS. O sobrenadante foi colhido para análise de citocinas por ELISA, e analisamos os polimorfismos nos genes de CD14 e TLR4 por PCR-RFLP. Não encontramos relação entre a exposição à endotoxina ambiental e o quadro de sibilância. Observamos que PBMC estimuladas ou não com LPS de crianças sibilantes e asmáticas produzem baixos níveis de IL-12 e IFN-<font face=\"Symbol\">&#947 quando comparado com crianças não sibilantes e não asmáticas. Os polimorfismos de TLR4 e CD14 não tiveram associação com sibilância ou asma. Nossos dados sugerem que não somente a polarização Th2 é importante para desenvolver essas patogenias, mas também uma diminuição na resposta Th1. / Although the great advance in the study of asthma and wheezing, there are numerous controversies about the involvement of endotoxin exposure, genetic background and cellular activation. We investigated the involvement of environmental endotoxin exposure and the role of LPS in the development of phenotypes wheezing and asthma. For experiments we selected wheezing and non-wheezing, and asthmatic and non-asthmatic children, and their blood collected and the PBMC cultured with LPS. The supernatant was collected for analysis cytokines by ELISA, and analyzed CD14 and TLR4 polymorphisms by PCR-RFLP. There was no relationship between environmental endotoxin exposure and the framework of wheezing. We observed that LPS-stimulated PBMC of wheezing and asthmatic children produce lower levels of IL-12 and IFN-<font face=\"Symbol\">&#947 when compared with non-wheezing and non-asthmatic children. The polymorphisms TLR4 and CD14 were not associated with wheezing or asthma. Our data suggest that not only Th2 polarization is important to develop these diseases, but also a decrease in Th1 response.

The goal of this research was to develop a novel biosensor for detecting and eliminating pathogenic E. coli. Traditionally, identifying pathogenic E. coli and distinguishing it from harmless environmental strains includes serotyping and DNA sequencing, which can take days or weeks. Our biosensor platform makes use of a material that is part of the immune system from single- multi- cellular organisms that target viruses, fungi, and bacteria called antimicrobial peptides (AMPs). Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we characterized non-specific binding between CP1 to silicon nitride and gold, and covalent binding of cysteine-terminated CP1 (CP1-cys) to gold. QCM-D monitors frequency and dissipative changes resulting from adsorbed mass, and peptide film thickness and density can be calculated using Voigt Viscoelastic modeling. Viability of the E. coli was monitored using a live/dead kit consisting of nucleic acid stains Syto 9 and Propidium Iodide. Successfully immobilizing peptide to a substrate is particularly important if CP1 would be applied on a food processing surface. By immobilizing CP1 to silicon nitride, we measured the binding forces between bacteria and peptides with the atomic force microscope (AFM), and explored important bacterial features such as LPS composition and length that influence binding affinity with CP1. The structure of the LPS is comprised of 3 sections: lipid A, core group, and O-antigen. We are mostly interested in the initial binding between AMP and LPS since our goal is to develop a novel biosensor that can detect pathogenic bacteria within seconds of exposure. Considering the short exposure period, the AMP would only be exposed to the O-antigen and outer core groups, which are repeating sugar chains that are essential for bacterial pathogenicity and adhesion to substrates. Although geared for use as a novel biosensor, results of this study can also be applied to the use of AMPs for replacing or enhancing the activity of antibiotics. Our work suggests that CP1 may not be serotype-specific, but targets the O-antigen before interfering with phospholipid groups of the bacterial membrane. Other factors that assist in pathogenicity, such as LPS length, may also be important for the consideration of CP1 potency.

Le genre Bordetella comporte à l’heure actuelle neuf espèces, majoritairement responsables d’infections respiratoires. B. pertussis, agent de la coqueluche, est le modèle de travail de cette thèse avec d’autres espèces telles que B. holmesii et B. avium. Les endotoxines bactériennes sont les composants essentiels de la membrane externe des bactéries à Gram-négatif. Du point de vue chimique, ce sont des lipopolysaccharides (LPS) qui provoquent un grand nombre de désordres physiopathologiques allant de la simple fièvre, à faible dose, jusqu’au choc endotoxinique mortel, à forte dose. L’analyse structurale des LPS de Bordetella est la spécialité majeure du laboratoire et la structure des endotoxines de la plupart des espèces de ce genre y a été décrite. Il est admis que le lipide A, qui constitue la région hydrophobe des LPS, est responsable de la majorité des activités biologiques de ces molécules. Ainsi, la moindre variation structurale de ces molécules a une répercussion importante sur la reconnaissance hôte-pathogène, les activités biologiques et la virulence bactérienne. A titre d’exemple, il a été mis en évidence que la modification spécifique, par substitution des groupes phosphate du lipide A, avec de la glucosamine, jouait un rôle majeur dans la modulation de la réponse immunitaire. Cette originalité structurale qui a été mise en évidence dans notre équipe chez B. avium, B. bronchiseptica puis chez B. pertussis; elle semble être un trait spécifique des Bordetelles. Il faut savoir que la coqueluche fait des ravages dans les pays sous-développés et touche les nouveau-nés dans de nombreux pays, comme la France, d’une maladie infectieuse mortelle dans les cas graves. Le vaccin qui ne peut être injecté qu’à l’âge de 2-3 mois et dont les rappels ne sont pas régulièrement suivis est imparfait. Les spécialistes du domaine ont reconnu qu’un complément antigénique serait nécessaire pour le rendre plus efficace. Au cours de cette thèse, nous avons analysé la structure des LPS d’isolats cliniques de B. pertussis afin d’étudier leur évolution et leur adaptation avec le temps ainsi que leur application vaccinale potentielle. De plus, concernant deux souches de B. pertussis, BP338 et BP18-323 nous avons contribué à la mise en évidence de nouveaux gènes impliqués dans la biosynthèse de la GlcN substituant les groupes phosphate de la région lipidique et à expliquer la différence de longueur du seul acide gras distinct entre les deux souches. L’étude de l’influence de ces éléments structuraux sur l’activation du complexe, TLR4/MD-2 apporte de nouveaux éclairages sur les interactions entre les lipides A et ce récepteur. Nos études sur les isolats cliniques de B. holmesii, pathogène opportuniste responsable d’affections de type coqueluche, montrent une grande hétérogénéité structurale du lipide A au sein d’un même isolat. Nous avons montré dans cette souche la présence d’un marqueur spécifique des souches de Bordetella, il s’agit d’un acide gras présent uniquement chez les lipides A des isolats humains. Nos travaux effectués sur des isolats cliniques de B. pertussis appartenant aux ères pré- et post-vaccinales et provenant de différents pays, montrent une perte du matériel génétique avec une déficience de certains antigènes majeurs. Nous avons démontré, via des méthodes physico-chimiques, que ces modifications ne concernaient pas les LPS de ces isolats. La stabilité de ces antigènes ainsi que nos méthodes de purification, nous permettent de proposer que ces LPS détoxifiés soient de bons candidats pour améliorer l’efficacité des vaccins coquelucheux acellulaires. Enfin, toutes les études structurales présentées dans cette thèse ont permis de mieux comprendre la régulation de certains gènes en réponse à un stress extérieur. Elles participent, sur l’exemple d’un pathogène majeur, au déchiffrage des mécanismes moléculaires qui mènent à la virulence et à l’adaptation bactérienne. / The Bordetella genus is actually composed of nine species responsible for respiratory infections. B. pertussis, the agent of whooping cough, is the main model of this thesis along with other species such as B. holmesii and B. avium. Bacterial endotoxins are the major components of Gram-negative bacteria external membrane. From a chemical point of view, they are lipopolysaccharides (LPS) causing a high number of pathophysiological disorders ranging from low fever at weak doses, to lethal endotoxic choc at high ones. Structural analysis of the Bordetellae LPS is the major specialty of our group where the endotoxin structures of most species of the genus were described. It is well-known that lipid A, which constitutes the hydrophobic moiety of LPS, is responsible for the majority of biological activities of these molecules. Thus, any structural change of these molecules has an important impact on host-pathogen recognition, biological activities and bacterial virulence. For example, it has been demonstrated that the specific modification by grafting glucosamine on lipid A phosphate groups plays a major role in modulating the immune response. This structural peculiarity was highlighted by our team first in B. avium, B. bronchiseptica then in B. pertussis; it seems to be a unique trait of Bordetella. It should be noted that pertussis wreaks havoc in developing countries and affects newborns in several others, including France, where this infectious disease causes a significant death toll. The vaccine, which cannot be injected before the age of 2-3 months, could be improved and boosters are not regularly monitored. Experts in the domain have recognized the lack of an antigenic complement to make it more effective. In this thesis, we analyzed the structure of LPS from B. pertussis clinical isolates to study their evolution and adaptation over time along with their potential use in the design of new vaccines. In addition, regarding two strains of B. pertussis, BP338 and BP18-323, we have contributed to the identification of new genes involved in the biosynthesis of GlcN substituting the phosphate groups of the lipid moiety, which helped explaining the difference in the length of the single fatty acid differing between the two strains. The analysis of the influence of these structural elements on the activation of the receptor complex, TLR4/MD-2 sheds new light on the interactions between lipids A and this receptor. Our studies on clinical isolates of B. holmesii, an opportunistic pathogen responsible for pertussis-like illness, show great structural heterogeneity in the lipid A of these isolates. We showed the presence of a specific marker of Bordetella species, namely a fatty acid present only in the lipid A of human isolates. Our works on B. pertussis clinical isolates belonging to pre- and post-vaccine eras and coming from different countries show a loss of genetic material with a deficiency in certain major antigens. We have demonstrated, via physico-chemical methods, that these modifications did not affect the LPS of these isolates. The stability of these antigens and our ability to purify them, allow us to propose that detoxified LPS could be good candidates for improving the effectiveness of acellular pertussis vaccines. Finally, all structural studies presented in this thesis have provided insight into the regulation of certain genes in response to external stress. Our compiled work on a major pathogen is an important step in deciphering the molecular mechanisms leading to bacterial virulence and adaptation.