Malária é uma das principais causas de doença e morte no mundo, principalmente de crianças. É considerada a força de seleção evolucionária mais forte que se conhece na história recente do genoma humano. Além dos fatores ambientais e do próprio parasito, fatores genéticos do hospedeiro têm um papel fundamental tanto na suscetibilidade como na evolução clínica da infecção. O sistema imune inato reconhece os plasmódios através de um número limitado de receptores de reconhecimento padrão (PRRs) e inicia vários mecanismos de defesa que resultam no desenvolvimento de inflamação e resistência do hospedeiro à infecção. Mas, a eliminação completa do parasito requer respostas imunes adaptativas que são amplificadas pela ativação do sistema imune inato. As manifestações clínicas de malária são dependentes dos níveis de citocinas próinflamatórias circulantes produzidas, as quais em níveis altos contribuem para a imunopatologia da doença. O balanço entre respostas pró e antiinflamatórias dirigidas contra o parasito é considerado crítico para a proteção clínica, assim a resposta imune inata pode contribuir tanto para proteção da malária como para modular a resposta imune adaptativa. Neste estudo, nós investigamos polimorfismos de um único nucleotídeo (SNP) dos genes de três PRRs: TLR, MBL e CR1 de indivíduos infectados por Plasmodium e residentes em áreas endêmicas de malária no Brasil. Os SNPs TLR1 (I602S), TLR4 (D229G), TLR6 (S249P), TLR9 (T-1237C/ -1486C), MBL [exon 1 nos códons 52, 54, e 57 (MBL2*A ou D, A ou B e A ou C, respectivamente); na região do promotor na posição -221 (*X ou *Y); e na posição +4 da região não traduzida (*P ou *Q)] e CR-1(C5507G) foram determinados por PCR-RFLP. Nós observamos associações entre os polimorfismos TLR1 I602S, TLR6 S249P e da região não traduzida +4 (*Q) e manifestações clínicas de malária e entre os polimorfismos TLR9 T-1486C, TLR T-1237C, MBL*D (códon 52) e do diplótipo de produção insuficiente de MBL (XA+O/O) e parasitemias mais altas. Nenhuma associação foi observada entre o polimorfismo CR-1 C5507G e manifestações clínicas de malária ou com parasitemia. Ao analisarmos juntos os polimorfismos de MBL e TLR, observamos que indivíduos com diplótipo de produção suficiente de MBL (YA/YA+YA/XA+YA/O+XA/XA) TLR1 I602S tinham menos manifestações clínicas de malária e indivíduos com diplótipo de produção suficiente de MBL e não carreadores do alelo TLR9 -1486C tinham parasitemias mais baixas do que os indivíduos com diplótipo de produção insuficiente de MBL e carreadores dos alelos variantes de TLR1 I602S e TLR9 -1486C, respectivamente. Juntos, nossos dados indicam que polimorfismos do promotor de TLR-9 e os diplótipos de produção insuficiente de MBL (XA+O/O) devem de algum modo controlar o nível de parasitemia por plasmódios enquanto a deficiência de TLR1 parece predispor para a presença de manifestações clínicas de malária. Também, podemos sugerir que existe uma cooperação entre TLR1, TLR9 e MBL na ativação da resposta imune inata na malária. Estes achados genéticos devem contribuir para o entendimento da patogênese da malária e levantar uma questão potencialmente interessante que é digna de investigações posteriores em outras populações a fim de validar a contribuição genética destes loci na patogênese da malária / Malaria is one of the major causes of disease and death worldwide, mainly of children. It is also the strongest known force for evolutionary selection in the recent history of the human genome. Besides environmental and parasite factors, host genetic factors play a major role in determining both susceptibility to malaria and the course of infection. Innate immune mechanisms directed against Plasmodium parasites both contribute to protection from malaria and modulate adaptive immune responses. The innate immune system recognizes Plasmodium via a limited number of pattern-recognition receptors (PRRs) and initiates a broad spectrum of defense mechanisms that result in the development of inflammation and host resistance to infection. But, the complete control of the infection requires adaptive immune responses; and the innate immune system is also very efficient in instructing the cellular mediators of adaptive immunity to lead a powerful additional strike force against the parasite. Clinical malaria is characterized by high levels of circulating proinflammatory cytokines, which are thought to contribute to the immunopathology of the disease. The balance between pro- and anti-inflammatory responses toward the parasite is considered critical for clinical protection. The innate immune system initiates and thus sets the threshold of immune responses. In this study, we investigated single nucleotide polymorphisms (SNP) in the genes of three PRRs: TLR, MBL and CR1 in Plasmodium-infected individuals living in endemic areas of Brazil. The SNPs TLR1 (I602S), TLR4 (D229G), TLR6 (S249P), TLR9 (T-1237C/ -1486C), MBL [in the coding sequence of exon 1 at codons 52, 54, and 57 (MBL2*A or D, A or B, and A or C, respectively); in the promoter region at position -221 (*X or *Y); and in the untranslated sequence at position +4 (*P or *Q)] and CR-1(C5507G) were determined by PCR-RFLP. We observed associations of the TLR1 I602S, TLR6 S249P and untranslated sequence at position +4 MBL (*Q) variants with clinical manifestations of malaria and of the TLR9 T-1486C, TLR9 T-1237C, MBL2*D and MBL-insufficient diplotype (XA+O/O) with higher parasitemias. No association was observed to the CR-1 C5507G ) and clinical manifestations of malaria or parasitemia. Also, we observed that individuals with MBLsufficient haplotype (YA/YA+YA/XA+YA/O+XA/XA) and not bearing the allele TLR1 I602S had less clinical manifestations of malaria and individuals with MBL-sufficient haplotype and not bearing TLR9 -1486C had lower parasitemias when compared to individuals with MBL-insufficient diplotype and bearing the variant alleles TLR1 I602S and TLR9 -1486C, respectively. Altogether, our data indicate that TLR-9 promoter and MBL-insufficient haplotype (XA+O/O) polymorphisms to some extent may control the level of Plasmodium parasitemia while TLR1 deficiency seems to predispose to mild malaria. Also, they could suggest cooperation among TLR1, TLR9 and MBL in the immune response against malaria. These genetic findings may contribute to the understanding of the pathogenesis of malaria and raise a potentially interesting issue that is worthy of further investigation in other population in order to validate the genetics contribution of these loci to the pathogenesis of malaria

Les effets anti-allergiques des bactéries lactiques sont suggérés par plusieurs études épidémiologiques, des essais cliniques et des modèles expérimentaux d’allergie. Cependant, les propriétés immunomodulatrices des bactéries lactiques sont sous-exploitées par les stratégies vaccinales développées pour combattre l’allergie et les mécanismes empruntés par ces bactéries pour moduler l’allergie restent peu caractérisés. Dès lors, nous avons caractérisé les propriétés immunomodulatrices qu’exerce Lactobacillus plantarum NCIMB8826, une bactérie lactique modèle, sur la cellule dendritique étant donné le rôle déterminant de cette cellule sur la réponse allergique. Nous montrons que L. plantarum induit une forte sécrétion d’IL-12 p40, d’IL-12 p70, de TNF-a mais une faible production d’IL-10. Cette faculté à induire la sécrétion de cytokines polarisantes dépend de TLR2, de TLR9, de MyD88, de NF-kB, des MAPKs (en particulier JNK, p38 et ERK 1/2), de la composition de l’acide lipotéichoïque de L. plantarum et de CD14. Nous montrons aussi que l’ADN génomique de L. plantarum est un agoniste de TLR9 et que CD14 et CD36 facilitent la liaison de la cellule dendritique avec L. plantarum. Ensuite, nous avons évalué le potentiel vaccinal d’une coadministration L. plantarum + Der p 1 dans un modèle murin d’allergie à Der p 1. Cette formulation vaccinale prévient la production d’IgE Der p 1-spécifique et atténue l’éosinophilie pulmonaire tout en stimulant une forte production d’anticorps IgG2a Der p 1-spécifiques et d’IFN-g par les cellules spléniques. Ces effets bénéfiques nous ont conduit à élaborer une bactérie lactique recombinante dérivée de L. plantarum produisant Der p 1 pour la vaccination contre l’allergie aux acariens. La forme antigénique que nous avons réussi à faire produire par L. plantarum correspond à une protéine de fusion entre la Maltose Binding Protein de E. coli et ProDer p 1 (le zymogène de Der p 1), la présence de ce partenaire de fusion étant indispensable à la production de ProDer p 1. En prophylaxie, la vaccination par cette bactérie recombinante prévient la production d’anticorps IgE-Der p 1-spécifiques et stimule la production d’anticorps IgG2a spécifiques, reproduisant les effets de la coadministration L. plantarum + Der p 1. Elle réduit de manière drastique la production d’IL-5 des cellules spléniques et des cellules ganglionnaires médiastinales et prévient l’éosinophilie pulmonaire mais n’a pas d’effet sur l’hyperréactivité bronchique. Der p 1 étant un des allergènes d’acarien les plus immunodominants, cet ensemble de données montre donc que cette bactérie recombinante constitue un vaccin prophylactique prometteur pour la prévention de l’allergie aux acariens. Des résultats préliminaires obtenus à partir de cellules dendritiques humaines et lymphocytes T autologues montrent la forte capacité de cette bactérie recombinante à induire le développement d’une réponse Th1 fortement polarisée (production d’IFN-g en l’absence de production d’IL-4 et d’IL-5), ce qui suggère que l’utilisation de cette bactérie recombinante pourrait être envisagée pour le traitement de l’allergie chez l’homme.

Allergic diseases are influenced by genetics and the environment. Maternal allergy appears to confer a higher risk for allergic sensitization than paternal allergy, suggesting an in utero influence. A decrease in particular infections or a lower exposure to microbial components during infancy is suggested to contribute to the high allergy prevalence in affluent societies. Toll-like receptors (TLR) 2 and 4 recognize peptidoglycan (PGN) and LPS respectively, are expressed on e.g. monocytes, and have been implicated in modulating the risk of IgE-sensitization. This thesis aimed to study the influence of maternal allergy and early microbial exposure on monocyte function and allergic sensitization during childhood. Blood samples from children participating in a prospective allergy cohort were used. Two-year old infants with allergic mothers had lower IL-6 production and reduced activation of the TLR-signalling intermediate p38-MAPK in response to PGN than children with non-allergic mothers. In 5-year old children, allergic disease and not maternal allergy influenced monocytic TLR2-regulation. Five-year olds who were seropositive for Epstein-Barr virus (EBV) at 2-years of age had a lower risk of persistent IgE-sensitization while EBV contraction after 2-years of age related to a higher risk of IgE-sensitization. Upon in vitro stimulation, NK cells from EBV+ 2-year olds produced lower IFN-g levels. EBV+ 2-year olds had also lower systemic IFN-g. In comparison to CD14++CD16- monocytes, CD14+CD16+ cells induced NK-cell IFN-g more potently in vitro, and EBV+ infants tended to have lower proportions of these CD14+CD16+ monocytes. This thesis highlights the importance of early-life microbial (EBV) exposure for a proper allergy-protective immunity. Also, maternal allergic heredity appears to influence monocytic microbial responses in early infancy. All these aspects relate to altered monocyte functionality, which suggest that they could have a role in allergic sensitization.

Atopic allergy is the most common chronic disease among children in the developed world. This high prevalence could be associated with low microbial exposure. The early gut microbiota appears to be important for immune maturation. Immunomodulatory components in human milk might differ between mothers and could therefore explain the contradictory results seen regarding breastfeeding and allergy development. The aim of this thesis was to investigate whether early colonization with certain gut microbiota species influences childhood immune responses and allergy development up to age five. Also, as human milk oligosaccharides (HMOs) might stimulate the growth of certain gut microbiota species, the consumption of neutral colostrum HMOs was investigated for their role in allergy development up to 18 months. The concentrations of neutral colostrum HMOs varied considerably between women; however this variation could not be explained by their allergic status. Neither was the consumption of neutral colostrum HMOs related to allergy development in their children up to 18 months. Infants who harboured lactobacilli group I and Bifidobacterium adolescentis one week after birth developed allergic disease less frequently during their first five years than infants who did not harbour these bacteria at the same time. Also, colonization with several Bifidobacterium species was associated with higher levels of house dust endotoxin and larger family size. The early Bifidobacterium flora influenced levels of salivary secretory IgA at six and 12 months but not during later childhood. Moreover, the intensity of early Bacteroides fragilis colonization was inversely associated with spontaneous Toll-like receptor 4 mRNA expression in peripheral blood cells collected 12 months after birth. In conclusion, these results indicate that the early infant gut microbiota influences systemic and mucosal immune maturation during infancy, and that it might be altered in infants developing allergic disease.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

Swine barn air is a heterogeneous mixture of dust, bacteria and irritant chemicals including ammonia and hydrogen sulphide. Gram-negative bacteria are commonly found in swine barn air and significantly contribute to pulmonary disease in unprotected swine barn workers, through the endotoxin moiety, lipopolysaccharide (LPS). Toll-like Receptor-4 is the ligand for LPS. It is found on many cell types including monocytes, macrophages, neutrophils, endothelial cells, and to a lesser extent, epithelial cells. The severity and outcome of acute lung injury following barn air exposures depends upon the balance between epithelial and vascular endothelial repair mechanisms, including angiogenesis. Vascular Endothelial Growth Factor (VEGF) is an endothelial mitogen produced by mesenchymal and alveolar Type II epithelial cells and by activated bronchial airway epithelial cells. Research investigating the role of cytokines in angiogenesis has shown that close proximity of immune cells and endothelial cells modulates the production of various compounds that regulate vascular function. Given that LPS is the ligand for TLR4 there appeared to be a role for TLR4 in angiogenesis, particularly following endotoxin exposure. To determine whether this was occurring, we examined whether exposure to swine barn air alters vascular density in the lungs and the role of TLR4 using a murine model. Toll-like Receptor-4 wild-type (C3HeB/FeJ) and TLR4 mutant (C3H/HeJ) mice were obtained and exposed to swine barn air for 1-, 5-, or 20-days for 8 hours/day. Wild-type animals showed a 127% increase in vascular density after 20-days barn air exposure. Vascular Endothelial Growth Factor-A protein levels were decreased by 0.62-fold after one-day swine barn air exposure in wild-type animals, indicating that VEGF-A is being used as a pro-angiogenic mitogen. Transcription of VEGF-A mRNA was increased in wild-type animals after all swine barn air exposure periods. The receptor VEGFR-1 showed increased mRNA transcription over all time points. These effects were only observed in TLR4 wild-type animals, indicating that these effects are mediated by TLR4. Further, VEGF-A and VEGFR-1 appear to be involved in the manifestation of TLR4-induced angiogenesis in the lung.

Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines. / text

Grâce aux nombreuses études sur le sujet, nous savons qu’une stimulation inflammatoire vasculaire excessive entraîne un débalancement des fonctions homéostatiques de l’endothélium. Ce débalancement est à l’origine d’une dysfonction endothéliale définie comme étant l’étape clé contribuant au développement de l’athérosclérose. Le Toll-like receptor-2 (TLR2) est impliqué dans l’activation cellulaire via la transcription des gènes liés à l’inflammation. Il reconnaît des molécules microbiennes mais également des facteurs endogènes non-infectieux tels que sécrétés par les tissus endommagés provenant de la dysfonction endothéliale. Ainsi, l’activation et la signalisation du TLR2 sont en étroite relation avec le développement de l’athérosclérose. Les études épidémiologiques ont confirmé le rôle athéroprotecteur de l’œstrogène via de nombreux mécanismes d’action. Ainsi, nous avons cherché à identifier de nouvelles cibles moléculaires permettant de mieux interpréter les bénéfices potentiels de l’œstrogène sur le système cardiaque. Pour la première fois chez les cellules endothéliales (CE) vasculaires de souris, nos travaux ont confirmé l’effet anti-inflammatoire de l’œstrogène via la diminution de l’expression et de l’activité du TLR2. Nous avons également déterminé l’influence de l’œstrogène sur le profil de la réponse inflammatoire de ce récepteur en mesurant les potentiels endothéliaux de migration et d’adhésion. De plus, nous avons caractérisé les voies de signalisation impliquées en démontrant l’influence négative de l’œstrogène sur la phosphorylation des kinases activées par le TLR2; illustrant l’interaction entre l’œstrogène et la signalisation de ce récepteur. Nos travaux amènent ainsi de nouvelles connaissances sur la régulation endothéliale du TLR2 et mettent en lumière les effets anti-inflammatoires et vasculaires rapides de l’œstrogène. / Evidence supports the contribution of immune responses in atherosclerosis development in part by alterations in the endothelium activation status and by the recruitment of inflammatory cells triggered by cardiovascular risk factors. These alterations are the principal cause of endothelial dysfunction defined as the key step contributing to the development of atherosclerosis. Via the transcription of genes related to inflammation, the Toll-like receptor-2 (TLR2) is involved in endothelial cell activation. It generally recognizes microbial molecules but also non-infectious endogenous factors such as those secreted by damaged tissues from the endothelial dysfunction. Thus, activation and signalization of the TLR2 are closely linked with the development of atherosclerosis. Epidemiological studies have confirmed the atheroprotective role of estrogen through multiple mechanisms of action. Thus, to better interpret the potential benefits of estrogen on the cardiovascular system, we sought to identify new molecular targets such as TLR2 regulation. For the first time in mouse vascular endothelial cells (EC), our results have confirmed the anti-inflammatory effect of estrogen via the decreased expression and activity of TLR2. We also determined the influence of estrogen on the profile of the inflammatory response triggered through this receptor by measuring endothelial migration and adhesion potentials. Furthermore, we demonstrated the interaction between estrogen and TLR2 signalling pathways with a negative influence of estrogen on the phosphorylation level of kinases activated by this receptor. Thus, our study brings new insights into the endothelial regulation of TLR2 and highlights rapid anti-inflammatory and cardioprotective effects from estrogen.

Die Gliazellen des enterischen Nervensystems galten lange Zeit lediglich als Packmaterial der Neuronen. Erst in den letzten Jahren rückte dieser Zelltyp in das Interesse der Forschung, weil mehrere Studien eine Schlüsselrolle bei der Erhaltung der Darmwandintegrität postulierten. Da bisher wenig über die immunphysiologischen Eigenschaften der enterischen Gliazellen bekannt war, wurde in dieser Arbeit deren Bedeutung bei der angeborenen intestinalen Immunantwort untersucht. Hierfür wurden Primärkulturen von enterischen Gliazellen eingesetzt, welche aus dem Plexus myentericus adulter Ratten stammten. Es wurde erstmalig nachgewiesen, dass enterische Gliazellen über Toll like und NOD-Rezeptoren (TLR 2, TLR 4, TLR 6, TLR 7, TLR 9, NOD 1, NOD 2) pathogene bakterielle Muster (PAMPs) erkennen. Dabei zeigte sich, dass eine enge Vernetzung zwischen den Toll-like-Rezeptoren besteht. Einerseits findet eine Liganden-spezifische Regulierung der Toll-like-Rezeptoren statt, anderseits beeinflussen TLR spezifische Liganden die mRNA-Expression weiterer Toll-like-Rezeptoren. In der Analyse der intrazellulären Signalweiterleitung konnte die Existenz verschiedener Adaptor- und Interaktionsmoleküle wie RICK und Myd88 sowie der IKK / NF κB Signalweg mit Degradierung von IκB α nach Aktivierung durch LPS belegt werden. Aktivierte enterische Gliazellen sind eine Quelle für pro- und anti-inflammatorische Interleukine (IL-1α, IL 1β, IL 6, IL-10, IL-12), TNF α und Chemokine (Ccl-2, Cxcl-9, Cxcl-10). Sie können somit weitere immunkompetente Zellen rekrutieren und agieren vermutlich als Schnittstelle zwischen der angeborenen und erworbenen Immunantwort. Die Ergebnisse dieser Arbeit implizieren, dass es sich bei enterischen Gliazellen um einen zusätzlichen immunregulatorischen Zelltyp im Darm handelt, welcher aktiv bei der angeborenen Immunabwehr mitwirkt. Neben pro inflammatorischen Eigenschaften tragen enterische Gliazellen auch zur Darmprotektion während Entzündungsprozessen bei. Daraus ergibt sich eine komplexe Funktionalität der enterischen Gliazellen bei der immunologischen Homöostase im Darm.

This work investigates the mechanisms of binding of T cell receptors (TCRs) to Class I MHC-peptide complexes (pMHC). The structure of a TCR specific for the Melan-A tumour antigen bound to its cognate pMHC was solved to a resolution of 2.5 Å which gives insight into how this TCR could be mutated to optimize binding and subsequently used as a cancer vaccine. Detailed sequence and geometric analyses of all currently available structures of Class I TCR-pMHC complexes revealed that TCRs can bind to pMHC with a range of orientations, yet always focus on the central portion of the peptide and use a specific subset of six residues on the MHC helices for binding. The most striking finding was the use of aromatic residues in the TCR CDR loops to bind to residue Q155 on the MHC α2 helix. Attempts were also made to express and purify Toll-like receptors (TLRs) with the aim of solving one or more of these structures. However, despite testing of over 50 different constructs from 12 different TLRs or associated proteins, insufficient soluble protein expression was obtained for crystallization trials. Finally, a protein disorder prediction tool was developed to aid construct design for structural biology studies and improve the chances of obtaining protein crystals. This tool is based on a novel type of neural network and blind tests comparing it to 8 other disorder prediction tools showed it is one of the best in the field. It is freely available at www.strubi.ox.ac.uk/RONN. Analysis of large datasets revealed that the position of order/disorder transitions is quite precisely defined in amino-acid sequences and that transition regions have an amino acid composition distinct from that of bulk ordered and disordered sequences. There is a steady decrease in order-promoting residues on the ordered side of boundaries as well as a weak sequence signal, both of which signify the approaching disorder and may prove useful for improving existing disorder prediction tools.