A contaminação da água para consumo humano por toxinas produzidas por cianobactérias é um problema de saúde pública e das autoridades em todo o mundo. Microcistina-LR (MCLR) é uma cianotoxina heptapeptídica cíclica que inibe as proteínas fosfatases PP1 E PP2A nos hepatócitos. Microcistinas são produzidas por diversos gêneros de cianobactérias e mais de 70 variações estruturais têm sido caracterizadas em florações naturais. Por serem haptenos, as microcistinas são incapazes de induzir uma resposta imune em animais. Conseqüentemente, foi necessário aplicar métodos de conjugação envolvendo a adição de uma proteína carreadora, mcKLH (cationized Keyhole Limpet Hemocyanin). Portanto, o objetivo inicial desta tese foi o de obter anticorpos monoclonal (em camundongos) e policlonal (em coelho) anti- MCLR. Com relação ao anticorpo monoclonal foram obtidos 9 hibridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232), sendo que apenas 5 se mostraram estáveis (k29, k317, k248, k284, k2232). Estes foram selecionados para serem isotipados, expandidos em líquido ascítico, purificados em coluna cromatográfica de proteína-A e titulados. Dentre estes cinco hibridomas secretores de anticorpos, o clone k317 foi o que melhor reconheceu (mais específico) a toxina MCLR. Os anticorpos do sobrenadante de meio de cultura do hibridoma e o fluido ascítico purificado foram identificados pelo ensaio ELISA (Enzyme Linked Immunosorbent Assay) previamente padronizado. Mesmo sensibilizando a placa de ELISA com diferentes antígenos, tais como MCLR-cBSA, MCLR, MCLR, MCRR, MCYR e MCLA, o clone 17 foi o que apresentou melhor linearidade frente às variantes de microcistina. Portanto, o clone 17 (isótipo IgG1) obtido é muito promissor e será usado para detecção de MCLR na água para consumo humano através do desenvolvimento de um kit de ELISA competição. Com relação ao anticorpo policlonal, o antígeno de imunização foi MCLR-mcKLH, enquanto que o antígeno de sensibilização foi MCLR-cBSA para o ensaio de titulação de anticorpos de classe IgG por ELISA indireto. Na seqüencia, foi padronizado um ensaio ELISA competição utilizando somente a toxina MCLR como antígeno de sensibilização. Este método Caseína foi padronizado, validado e comparado com o kit comercial Abraxis®. O kit ELISA competição que utiliza anticorpo policlonal, nomeado como método Caseína, foi avaliado quanto Limite Inferior de Quantificação, Especificidade, Seletividade, influência do metanol no ensaio, Recuperação, Linearidade, Precisão, Exatidão e Robustez. Este método de triagem apresentou excelente resultado quando comparado ao kit comercial Abraxis®, pois foi capaz de detectar tanto variantes de microcistinas como nodularinas no ambiente aquático. O ensaio ELISA competição utilizando anticorpo policlonal anti-MCLR foi submetido à patente pela Agência USP de Inovação (I.N.P.I. 018090046230). / The contamination of drinking water by cyanobacterial toxins is a public health issue and a concern for water authorities throughout the world. Microcystin-LR (MCLR) is a hazardous cyclic heptapeptide cyanotoxin, which inhibits protein phosphatase PP1 and PP2A in hepatocytes. Microcystins are produced by several genera of cyanobacteria and presents more than 70 structural variations characterized in natural blooms. As haptens, microcystins are unable to invoke an immune response in animals. Consequently, the application of conjugation methods with an additional carrier protein, the KLH (Keyhole Limpet Hemocyanin) was necessary. The main objective of this study was to obtain monoclonal (in mice) and polyclonal (in rabbits) antibodies for reacting against MCLR. In what refers to monoclonal antibodies, 9 hybridomas (k29, k210, k317, k248, k284, k290, k2161, k2226, k2232) were obtained; however only 5 were stables (k29, k317, k248, k284, k2232). These were selected to be isotyped, expanded in ascitic fluid, purified by protein-A column chromatography and then, they were titrated. Out of these five antibody-secretor hybridomas, clone k317 was the best to recognize (more specific) the MCLR toxins. Antibodies in hybridoma cell culture supernatant and purified ascites fluid were identified by ELISA assay (Enzyme Linked Immunosorbent Assay) as prior standardized. Even when sensitizing ELISA plate with different antigens, as MCLR-cBSA, MCLR, MCLR, MCRR, MCYR and MCLA, clone 17 presented the best linearity against microcystin variants. Therefore, the obtained clone 17 (isotype IgG1) is a promising clone and shall be used for detecting MCLR in drinking water through the development of a competitive ELISA immunoassay kit. In what refers to the polyclonal antibody, MCLR-mcKLH was used as immunization antigen, while MCLR-cBSA was used as sensitizing antigen for the IgG titration assay by indirect ELISA. In the sequence, a competition ELISA assay was standardized using the MCLR toxin as sensitizing antigen. This Casein method was standardized, validated and compared to the commercial kit Abraxis®. The competition ELISA kit using polyclonal antibody, known as Casein method, was analyzed concerning its Quantification Inferior Limit, Specificity, Selectivity, methanol influence of the assay, Recuperation, Linearity, Precision, Accuracy and Robustness. This screening method reached excellent results if compared to the commercial kit Abraxis®, for being able to detect both the microcystins variants and the nodularins in aquatic environmental. The competition ELISA assay using anti-MCLR polyclonal antibody was submitted to the grant of a patent by USP Innovation Agency (INPI 018090046230).

In this thesis molecular interactions between polypeptide conjugates and protein targets were investigated. Polypeptides were derivatized with small organic molecules, peptides and oligonucleotides. New strategies were developed with the aim to increase affinities for proteins of biological interest. A 42-residue polypeptide (4-C15L8) conjugated to a small organic molecule 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid (PP1), was shown to bind glycogen phosphorylase a (GPa) in the presence of zinc ions. Under the assumption that hydrophobic interactions dominated the binding energy, the hydrophobic residues of 4-C15L8-PP1 were systematically replaced in order to study their contribution to the affinity enhancement. The replacement of the Nle, Ile and Leu residues by Ala amino acids reduced affinities. The introduction of non-natural L-2-aminooctanoic acid (Aoc) residues into the peptide sequence enhanced the binding affinity for GPa. A decreased KD of 27nM was obtained when Nle5, Ile9 and Leu12 were replaced by Aoc residues, in comparison to the KD value of 280nM obtained for the unmodified 4-C15L8-PP1. It is evident that there are non-obvious hydrophobic binding sites on the surfaces of proteins that could be identified by introducing the more hydrophobic and conformationally flexible Aoc residues. The downsizing of the 42-mer peptide to an 11-mer and the incorporation of three Aoc residues gave rise to a KD of 550 nM, comparable to that of  4-C15L8-PP1 suggesting that bioactive peptides can be downsized by the introduction of Aoc. Aiming to improve in vivo stability, the affinity for human serum albumin (HSA) of hydrophobic, positively and negatively charged polypeptide-PP1 conjugates was evaluated. Increased hydrophobicity due to the introduction of Aoc residues did not significantly increase the affinity for HSA. No binding was observed in the case of the most negatively charged polypeptides whereas the slightly negatively and positively charged polypeptides conjugated to PP1 bound HSA with affinities that increased with the positive charge. It was found that polypeptide-PP1 conjugates target the zinc binding site of the HSA. Affinity enhancement was obtained due to the incorporation of PP1 and increased by charge to charge interactions between the positively charged amino acids of the polypeptide and the negatively charged residues of HSA, in close proximity to the HSA zinc binding site. The survival times of the peptide-PP1 conjugates in human serum were extended as a result of binding to HSA. Zn2+ ion chelating agents can be incorporated in potential peptide therapeutics with a short plasma half-life, without increasing their molecular weights.

Les infections à Pseudomonas aeruginosa sont un problème de santé publique important et peu de solutions thérapeutiques sont disponibles face à des souches isolées multi-résistantes. Le séquençage de souches de P. aeruginosa a montré qu'en plus du génome cœur, il existe de nombreux gènes accessoires. La souche PA14 est un isolat clinique hautement virulent, de part la présence de deux îlots de pathogénicité, PAPI-1 et PAPI-2, contribuant de manière individuelle et synergique à la virulence. L'îlot PAPI-1, de 108 kb, est un élément intégratif et conjugatif (ICE), capable de s'auto-transférer à des souches de Pseudomonas par un mécanisme de conjugaison. Le mécanisme de transfert fait intervenir un pilus de type IVb encodé dans l'îlot PAPI-1.Mon travail de thèse a eu pour objectif d'identifier les régulateurs de ce locus pilPAPI-1. Ce travail a montré que ce locus est faiblement exprimé mais qu'il peut être induit par l'ajout d'acide caprique. Par une approche de mutagénèse aléatoire, j'ai démontré qu'il existe au moins deux gènes de fonctions inconnues présents dans PAPI-1 nécessaires à l'activation du locus et au transfert de l'îlot : RL103, qui coderait pour un régulateur transcriptionnel de type Ribbon-Helix-Helix (RHH) et RL102, qui coderait pour une protéine de partitionnement chromosomique. Par une approche transcriptomique, j'ai démontré que ces deux régulateurs sont aussi impliqués dans l'activation de l'expression de plus de 35% des gènes de PAPI-1. Dans leur ensemble, ces résultats ont mis en évidence que RL103 et RL102 sont deux activateurs du transfert de PAPI-1 et ont montré le premier exemple de régulateur de type RHH impliqué dans le transfert d'un ICE. / P. aeruginosa infections have become a serious threat to public health and are very difficult to treat due to the increasingly emergence of strains resistant to all known antibiotics. Sequencing of P. aeruginosa strains showed, that in addition to a conserved core genome, there are variable accessory genes. The PA14 strain is a highly virulent clinical and this is mainly due to two pathogenicity islands, PAPI-1 and PAPI-2, that contribute individually and synergistically to the virulence. The 108 kb PAPI-1 pathogenicity island is an integrative and conjugative element (ICE), capable of self-transferring to any recipient Pseudomonas strain by a conjugative mechanism. The transfer mechanism is mediated by a type IVb pilus, encoded within the PAPI-1 island. My PhD work was aimed to identifying the regulators of this pilPAPI-1 locus. This work showed that this locus is weakly expressed but may be induced by the addition of capric acid. Using a random mutagenesis approach, i have shown that there are at least two genes of unknown function (present in PAPI-1) necessary for activation of the locus pilPAPI-1 and the transfer of the island : RL103, which would encode a Ribbon Helix Helix-like transcriptional regulator and RL102, which would encode a partitioning chromosome protein. Using a transcriptomic approach with microarrays, I demonstrated that these two regulators are also involved in the activation of the expression of more than 35% of PAPI-1 genes. Taken together, these results show that Cpr and RL102 are two activators of the PAPI-1 transfer of PA14 and show the first example of a Ribbon-Helix-Helix transcriptional regulator involved in the transfer of an ICE.

No description available.

Plasmids are small, circular, extrachromosomal double-stranded genetic elements present in bacteria. Plasmids can replicate independently of the bacterial chromosome and play an important role as a transmitter of antibiotic resistance genes between bacteria. Antibiotic resistance genes have been shown to be selected for even in the presence of subinhibitory levels of antibiotics, but the effect of antibiotics on conjugation is not as well understood. To study this, we designed a novel conjugation tracking system utilizing a Cre-expressing plasmid and a chromosomal floxed blue fluorescent protein (BFP) gene. We found that our model worked opposite as intended as cells expressed BFP before conjugation and lost BFP expression upon recombination. An issue with the system was isolated to the direction of the single loxP site remaining after recombination. Both loxP sites were inverted but this did not restore the intended expression of BFP after recombination. Subsequently the system was modified to increase the space between the promoter region and the single loxP site remaining after recombination. This extension produced the desired result as BFP expression now increased upon recombination. Still, further work needs to be done to construct a Cre-expressing plasmid, tune expression of BFP, and show expression of yellow fluorescent protein (YFP) in our model before the system can be applied to clinical isolates.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19406号 / 工博第4122号 / 新制||工||1635(附属図書館) / 32431 / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 中條 善樹, 教授 澤本 光男, 教授 赤木 和夫 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(工学) / 乙第13145号 / 論工博第4163号 / 新制||工||1687(附属図書館) / (主査)教授 森 泰生, 教授 浜地 格, 教授 梅田 眞郷 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

No description available.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.