Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%. / Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.

Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

Research into inorganic supramolecular chemistry is burgeoning, in particular that which focuses on the formation of capsular molecules and the effects that these unique environments have on catalytic reactions. With the aim of producing new ligand designs that could not only support reactive metals, but also partake in supramolecular aggregation to provide a capsular microenvironment, new tripodal ligands and wide span imines and amines have been synthesised. Furthermore, the exploitation of hydrogen-bonding motifs formed through pyrrole-imine tautomerisation upon metallation of these ligands has been explored, with the aim of enhancing reactivity and stabilising reactive intermediates. In Chapter one, the concept of covalent and non-covalent capsules is introduced, and includes the different aspects affecting the encapsulation of molecules and their use as nanoreactors. The use of secondary interactions, e.g. hydrogen-bonding in metal complexes of tetrapodal and tripodal ligands is discussed. Chapter two describes the synthesis of a tripodal pyrrole-imine ligand and the formation of its multi-metallic complexes of Group one metals, transition metal and the f-block elements. The complete and partial tautomerisation of this ligand upon metal complexation is also examined. In Chapter three, the formation of hangman complexes of the tripodal pyrrole-imine ligand is described and is extrapolated to the chemistry of a new pyrrole-amide ligand. The synthesis of this latter ligand and its properties with regards to anion binding are also explored. Chapter four describes the formation of wide span diamine and diimine ligands and their propensity to form adducts with cobalt and zinc chlorometallates and unusual multimetallic palladium complexes. The final conclusions of the work presented in this thesis are drawn in Chapter five. Chapter six presents experimental details and characterising data for all of the new compounds presented in this thesis.

Photobacterium profundum SS9 is a γ-proteobacterium which grows optimally at 15°C and 28 MPa (a psychrophilic piezophile) and can grow over a range of temperatures (2-20oC) and pressures (0.1-90 MPa). Previous research had demonstrated that P. profundum SS9 adapts its membrane proteins and phospholipids in response to growth conditions. In this study, methodology was developed for growing P. profundum SS9 under cold temperatures and high pressures in both liquid and solid cultures. The effect of changing growth conditions on cell envelope polysaccharides was then investigated. The lipopolysaccharide (LPS) profile of a rifampicin resistant P. profundum SS9 derivative, SS9R, was shown to change at 0.1 MPa with respect to temperature and at 15°C with respect to pressure. Compositional analysis showed that the LPS was almost entirely composed of glucose. This provides evidence that, under these conditions, the major polysaccharide produced by P. profundum SS9 is a glucan. Two putative polysaccharide mutants, FL26 & FL9, were previously isolated from a screen for cold-sensitive mutants of P. profundum SS9R. Both mutants displayed an increased sensitivity to cold temperatures on solid medium and were unaffected in their growth at high pressure. FL26 was found to exhibit an LPS alteration similar to previously published O-antigen ligase mutants, providing evidence that this mutant is likely to lack O-antigen ligase. Interestingly, FL26 was also shown to have a reduced ability to form biofilms and had increased swimming motility. This suggests that there are a number of changes which occur in FL26 in the absence of O-antigen. FL9 was found to have an altered LPS and capsular polysaccharide (CPS), similar to an E. coli wzc mutant. In E. coli, Wzc is involved in the polymerisation and transport of CPS, disruption of which can also lead to LPS alterations. The LPS and CPS alterations may lead to the cold-sensitivity phenotype, either individually or in combination. In conclusion, alterations in the cell envelope polysaccharides were shown to affect cold temperature sensitivity on solid agar. Cold-sensitivity is most likely directly related to the LPS alterations and stability of the membrane under cold temperatures. Exopolysaccharides (EPS) have previously been shown to affect desiccation and freezethaw resistance, making it is possible that the CPS plays a similar role in this case.

Burkholderia pseudomallei is the aetiological agent of melioidosis and a potential bioterror threat. Infections are difficult to treat due to extensive antibiotic resistance and there is no prophylactic vaccine available. Studies have shown that the capsular polysaccharide (CPS) of B. pseudomallei is a virulence factor, immunogen and candidate antigen for a glycoconjugate vaccine. However, polysaccharides are complex to synthesise. One approach is to genetically engineer Escherichia coli to express the CPS; however, previous attempts at cloning the CPS coding locus from B. pseudomallei into E. coli were unsuccessful. This project proposes to clone only the essential genes from B. pseudomallei and to use native E. coli mechanisms to complete CPS synthesis. This would contribute to development of a new platform for the expression of any bespoke polysaccharide in E. coli. Six biosynthetic genes for the nucleotide sugar precursor were successfully expressed in E. coli. The structure of the precursor was verified by mass spectrometry. Precursor synthesis was also performed in an in vitro microfluidics system. This minimised the quantity of substrates and enzymes required, in preparation for the characterisation of glycosyltransferases required for CPS assembly. A novel assay for characterising glycosyltransferase activity was also developed, as current available options are prohibitively expensive and require significant quantities of glycosyltransferase which are difficult to purify. Finally, plasmids for the expression of additional glycosyltransferases to link the nascent B. pseudomallei CPS to truncated polysaccharides in E. coli were constructed. The aim of this project was to contribute to the development of a platform for the expression of bespoke polysaccharides in E. coli. The CPS of B. pseudomallei was chosen as the model polysaccharide as it has a simple structure and its manufacture is desirable for use in a vaccine against melioidosis.

A thesis by Marc Ashley Bayliss entitled ‘Virus-like particles as a novel platform for delivery of protective Burkholderia antigens’ and submitted to the University of Exeter for the degree of Doctor of Philosophy. There is currently no licensed vaccine available for the global tropical pathogen Burkholderia pseudomallei which is the causative agent of melioidosis and a potential bio-threat agent. The capsule polysaccharide (CPS) expressed by B. pseudomallei has been shown to offer some protection against bacterial challenge. Polysaccharide immunogenicity can be enhanced by conjugation to a carrier protein and several licensed vaccines utilise this technology. Virus-like particles (VLPs) are non-infectious, non-replicating, viral proteins that self-assemble into viral structures and are in several licensed vaccines as primary antigens. VLPs are also effective delivery platforms for foreign antigens by genetic insertion or chemical conjugation. iQur, a collaborator on this project, has developed Tandem CoreTM that consists of two genetically linked hepatitis B core proteins that allow insertion of large proteins into each core whilst remaining assembly competent. The aim of this thesis was to assess the protective efficacy of Tandem CoreTM VLPs chemically conjugated to CPS and Tandem CoreTM Burkholderia protein fusion constructs. This involved three objectives; reduce the cost of CPS extraction; identify immunogenic Burkholderia proteins; and test candidate vaccine efficacy in an animal model of acute melioidosis against B. pseudomallei challenge. To reduce the cost of extraction, CPS was purified from B. thailandensis strain E555 and bacterial culture CPS concentration optimised which first required development of a quantitative ELISA. Immunogenic Burkholderia proteins were identified from the literature but Tandem CoreTM fusion constructs containing these proteins were not assembly competent. The Burkholderia proteins were added as co-antigens to the VLP CPS conjugate vaccine but did not improve efficacy. Tandem CoreTM VLPs conjugated to CPS were protective against B. pseudomallei challenge and were compared to CPS conjugated to Crm197: a commercially available carrier protein used in several licensed vaccines. At lower challenge doses, survival was greater in mice vaccinated with the VLP-CPS conjugate although at higher doses, Crm197-CPS efficacy was greater.

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No description available.

La réalisation d'une transformation chimique dans un espace confiné constitue pour les chimistes molécularistes un véritable défi. Des recherches récentes ont montré que des réactions se déroulant dans une poche ou une cavité moléculaire étaient de nature à engendrer des sélectivités nouvelles et faciliter des réactions thermodynamiquement défavorables. L'association métal-cavité permet également le déroulement de processus catalytiques en milieu aqueux, dès lors que la cavité a été rendue hydrosoluble.L’objectif de cette thèse était de préparer des ligands originaux intégrant une ou plusieurs cavités moléculaires de type calix[4]arène et d’en étudier les propriétés complexantes. L’ensemble des calixarènes synthétisés sont porteurs d’un ou plusieurs groupes PPh2 directement liés au bord supérieur du macrocycle. Plusieurs types de molécules ont été préparées : (i) des bis-calixarènes formant, après complexation, des métallo-capsules; (ii) des calixar!ène-diphosphines adaptées à la formation de complexes bimétalliques dans lesquels les centres métalliques sont placés entre deux coquilles se faisant face. L’activité catalytique de certains de ces métallo-capsules est jusqu’à 40 fois supérieurs à celle observé pour un catalyseur classique.La dernière partie de cette thèse a pour objectif de mettre à la disposition des spécialistes de la chimie des surfaces des phosphacalixarènes originaux destinés à la confection de supports solides P(III)-fonctionnalisés et donc de nouveaux catalyseurs supportés. / The realization of a chemical transformation in a confined space is for molecularist chemists a challenge. Recent research has shown that reactions occurring in a pocket or a molecular cavity were such as to generate selectivities and facilitate new thermodynamically unfavorable reactions.The metal-cavity association allows also the course of catalytic processes in aqueous medium, when the cavity has been made water soluble.The objective of this thesis was to prepare original ligands incorporating one or more cavities of molecular type calix[4]arene and to study their complexing properties. All the synthesized calixarenes are carriers of one or more groups PPh2 directly related to the upper rim of the macrocycle.Several types of molecules were prepared: (i) bis-calixarenes forming, after complexation, metallocapsules, (ii) calixarene-diphosphines suitable for the formation of bimetallic complexes in which the metal centers between two shells are placed facing each other. The catalytic activity of some of these metallo-capsules is 40 times higher than that observed for a conventional catalyst.The last part of this thesis aims to make available experts in surface chemistry of the original phosphacalixarenes for making solid supports P (III)-functionalized and thus new supported catalysts