Dans l’optique de développer de nouveaux agents bio-inspirés pour la détection et/ou le traitement des cellules cancéreuses, nos travaux se sont tournés vers la synthèse de macromolécules peptidiques complexes ayant la capacité de reconnaître les cellules tumorales. Ces travaux visent à développer des molécules permettant de cibler des particularités cellulaires présentes sur les cellules tumorales dans le but d’obtenir un traitement personnalisé via une vectorisation active permettant une augmentation de l'efficacité thérapeutique et une réduction intrinsèque de la toxicité du traitement. Pour cela, ces biomolécules doivent posséder à la fois un site de reconnaissance pour la liaison avec des protéines présentes à la surface de la cellule cible et un ou plusieurs éléments utilisés pour détecter et/ou détruire la cible. Ces systèmes ont été élaborés à partir d'un châssis moléculaire cyclodécapeptidique présentant des propriétés conformationnelles particulières. Plusieurs approches ont été envisagées. La première a consisté à rechercher de nouveaux ligands de récepteurs tumoraux en s'inspirant du domaine de reconnaissance d'un anticorps monoclonal thérapeutique. Dans ce contexte, nous avons proposé la conception de mimes du Rituximab ciblant l'antigène CD20 utilisé dans le traitement des lymphomes Non-Hodgkinien. Dans la seconde approche, nous avons développé des vecteurs destinés à des applications d'imagerie tumorale. Pour cela, des châssis multivalents présentant des ligands peptidiques RGD ciblant l'intégrine alpha-v-beta-3 ont été conjugués avec différents agents de détection puis évalués par des techniques d'imagerie telles que la TEP, la TEMP et l’imagerie optique. Toujours dans un but de diagnostic des cellules tumorales, nous nous sommes par la suite tournés vers l’application à la capture cellulaire. Pour cela, une surface d’or à été modifiée via la formation d’une monocouche organisée SAM (« Self-assembled monolayer ») présentant des cyclodextrines. Un gabarit peptidique adéquat a ainsi permis la capture et le relargage sélectif de cellules tumorales mesurées par la technique de microbalance à quartz. Ces mêmes vecteurs, validés pour le diagnostic ont par la suite été couplés à des peptides cytotoxiques issus d’une protéine pro-apoptotique « Bax ». Enfin, une dernière partie a été consacrée à la recherche de nouveaux composés comportant plusieurs éléments de ciblage tumoraux. Ces molécules présentent deux ligands de ciblage des récepteurs surexprimés sur la membrane et peuvent ainsi permettre une meilleure sélectivité vis-à-vis des tissus tumoraux. / In order to develop new agents for cancer diagnosis and treatment, our work aims to synthesize complex peptide macromolecules that are able to specifically recognize cancer cells. Our goal is to increase the therapeutic efficiency and reduce the toxicity of currently available drugs using "targeted strategies". In this context, we designed sophisticated macromolecules encompassing a cell recognition domain and one or several components used to detect and/or destroy the target. This system was prepared starting from a cyclodecapeptidic scaffold presenting particular conformational properties. Different approaches were considered. First of all our work was to investigate new tumor receptor ligands based on the recognition domain of a therapeutics monoclonal antibody. We proposed the design of Rituximab mimics which targets the CD20 antigen used for the treatment of Non-Hodgkin lymphoma. In a second approach, we prepared new vectors for tumor imaging. For this purpose, multivalent scaffolds containing RGD peptide that targets alpha-v-beta-3 integrin were combined with several detection elements and evaluated by using PET, SPECT and optical imaging techniques. We also used this peptide vector for the selective cell capture and release from flowing suspensions, using a gold surface modified with a cyclodextrin-containing self-assembled monolayer (SAM). A scaffold containing ferrocenyl and -RGD- ligands permitted the selective capture and release of tumor cells. This experiment was monitored by QCM-D. This vector has been next grafted to a cytotoxic peptide that was discovered from a pro-apoptotic protein named “Bax”. Finally, we designed new molecules which include an additional ligand for the cell’s surface to increase the selectivity and the affinity of tumor tissue.

Câncer é a denominação para um grupo de doenças devastadoras caracterizadas pelo crescimento e multiplicação descontrolados de células anormais que são capazes de invadir estruturas próximas e se espalhar por diversas regiões do organismo.Trata-se de um grande problema de saúde pública mundial, fazendo milhares de novas vítimas a cada ano. De acordo com o Instituto Nacional do Câncer (INCA), são estimados cerca de 580 mil casos novos da doença no Brasil para 2014. A presente tese de doutorado teve como foco principal a identificação e o desenvolvimento de novas moléculas com atividade anticâncer, através de triagens biológicas de compostos de origem natural e sintética, e do estudo das relações entre a estrutura e atividade (SAR). Triagens in vitro de derivados sintéticos do ácido gálico, ácido protocatecuico e guanidínicos, além de uma variedade de produtos naturais, possibilitaram a identificação de agentes inibidores da migração e proliferação de células tumorais metastáticas. Uma série de derivados sintéticos indólicos e espirocicloexadienonas, com potente efeito inibitório da migração celular, teve caracterizada a sua ação frente à proteína tubulina, alvo molecular de compostos importantes como o taxol, a vimblastina e a colchicina. Ensaios de imunofluorescência revelaram a ação dos compostos associada a alterações do citoesqueleto celular. Também foram realizados o planejamento e a síntese de três cadeias peptídicas por meio da metodologia de síntese peptídica em fase sólida. Os resultados da avaliação biológica dos peptídeos indicaram o efeito de inibição na migração e proliferação de células tumorais de mama. Finalmente, estudos de metabolômica de duas linhagens tumorais de mama foram conduzidos através de análises de RMN do material celular cultivado. / Cancer is a group of devastating diseases characterized by the abnormal growth of defective cells which invade adjacent tissues and eventually disseminate to several locations of the body. It is a major public health problem, affecting thousands of people each year. According to the brazilian National Institute of Cancer (INCA), approximately 580,000 new cases of cancer are predicted for the year of 2014 in Brazil. The main goal of this PhD thesis was the identification and development of new molecules possessing anticancer activity, through biological screenings of compounds from natural and synthetic sources, as well as the investigation of structure-activity relationships (SAR). In vitro screening of synthetic derivatives of gallic acid, protocatechuic acid and guanidines, along with a diverse set of natural products, allowed the identification of inhibitors of cellular migration and metastatic cell proliferation. A series of synthetic indolic derivatives and cyclohexanediones, having potent inhibitory activity in cellular migration, was characterized upon tubulin, an important macromolecular target for compounds such as taxol, vinblastine and colchicine. Immunofluorescence assays revealed that the compounds act by altering the cellular cytoskeleton. The design and synthesis of three polypeptides were also performed through solid phase synthesis. The biological evaluation of the peptides demonstrated their inhibition effects on the migration and proliferation of breast cancer cells. Finally, metabolomic studies of two strains of breast cancer cells were conducted by NMR analyses of cellular cultures.

Microarrays on gold have been used to develop new methodologies for biocatalysis and proteomic applications. The technology applies the logic of solid phase supported chemistry using self-assembled monolayers (SAMs) on a gold chip. The advantages of this technology are: i) an easy to handle platform, ii) parallel screening (64 reactions at once), iii) microliter scale reactions (1µL per sample), iv the use of mild conditions (buffers and t=37 °C), v) the absence of purification steps (only chip washing is required), vi) quick and accurate analysis by MALDI ToF MS.The metallopeptidase thermolysin was studied in peptide-coupling reactions to profile reactivity and specificity (Chapter 2). Reactivity was further investigated in transpeptidation reactions. Comparing the serine peptidase chymotrypsin with the zinc dependent thermolysin, it was found that transpeptidations proceed via N-transacylation reactions independent of a specific enzymatic catalytic mechanism (Chapter 3). These transacylations may be exploited for modifications of biocompatible and selective surfaces in ‘bottom-up’ bionanofabrication technologies. Selected peptidases with different catalytic mechanism were also arrayed to investigate polymerisation ability of dipeptides (Chapter 4). It was shown that oligomerisation can be obtained under mild conditions and a set of peptides was synthesised. Chapter 5 describes a new chemical methodology by which crude tryptic peptide digests can be trapped on chip and analysed by MALDI ToF MS without further purification steps. This dramatically improves time and cost efficiency.Finally, a new stepwise native chemical ligation methodology is proposed for amino acids, and peptides containing N-terminal cysteine residues (Chapter 6).

Plant signaling involves the transport of information within and between plant cells from receptors to effectors. Plants are affected by biotic and abiotic stress conditions like insect attack and extreme temperatures, respectively, which cause disease, the induction of senescence and the reduction of crop yield. To improve plant traits for feed, fiber, and energy applications, it is critical to understand the short- and long-range signaling mechanisms plants use to control growth, biomass composition, senescence and responses to environmental stresses. It is known that many plant signaling molecules have profound effects on plants, through mechanisms that remain largely obscure. A key gap in knowledge is the understanding of the mechanisms that govern the movement and fate of signaling molecules. This study seeks to synthesize signaling probes based on flagellin 22 (flg22), a 22-amino acid peptide that induces defense gene expression to trigger both local and systemic immune responses in plants. Solid-phase synthesis of fluorescently-tagged derivatives of flg22 was initiated, and studies on the uptake of labeled probes was conducted using a fiber-optic fluorescence microscope that was adapted for use in plants. Fluorescence microscopy showed uptake and internalization of TAMRA-flg22 in cells of Arabidopsis thaliana Columbia (wild-type strain), which was not observed in the fls2 strain in which FLS2, the receptor for flg22, had been knocked out.

Orientador: Eduardo Maffud Cilli / Banca: Eduardo Maffud Cilli / Banca: Clovis Ryuichi Nakaie / Banca: Hosana Maria Debonsi Navickiene / Resumo: Este trabalho descreve a sintese, a relacao estrutura-funcao, a interacao com membranas e a atividade biologica de um peptideo proveniente da regiao amino-terminal da proteina Esticolisina II (St II). Esta proteina e uma citolisina pertencente a familia das actinoporinas, obtida da anemona marinha Stichodactyla heliantus. Estudos estruturais e biologicos da proteina nativa indicam que a regiao amino-terminal da St II participa do processo de formacao de poros em membranas. Deste modo, visando compreender o comportamento e a importancia dessa regiao para a atividade biologica da St II, 3 analogos contendo os 30 primeiros residuos que compoem a extremidade amino-terminal da St II foram sintetizados e analisados. Os peptideos foram obtidos mediante sintese em fase solida modificando-se um residuo de leucina ou isoleucina por triptofano nas posicoes 2, 7 ou 24. Os estudos conformacionais foram realizados atraves das tecnicas de fluorescencia e dicroismo circular. Para avaliar o comportamento estrutural dos peptideos em solucao, foram realizados estudos de variacao de pH, forca ionica e titulacao com um agente indutor de estrutura, o trifluoroetanol (TFE). Na interacao com mimeticos de membranas, os peptideos foram titulados por tres tipos de detergentes: dodecil-sulfato de sodio (SDS), HPS (N-hexadecil-N,N-dimetil-3-amonio-1-propano-sulfonato) e LPC (1-palmitoil-2-hidroxi-sn-glicero-3- fosfocolina). Complementando o estudo acima, tambem foram realizados experimentos utilizando acrilamida como agente supressor de fluorescencia. Os resultados obtidos demonstram que a estrategia de sintese de peptideos em fase solida atraves do protocolo Fmoc/tBu, utilizada na sintese dos peptideos deste trabalho, foi viavel. O processo de purificacao dos peptideos atraves de HPLC tambem se mostrou eficiente e viabilizou a obtencao do material com alto indice de pureza. / Abstract: In this study, we describe the synthesis, the relation function-structure, the interaction with membranes and the biological activity of the N-terminus region of the protein Sticholysin II (St II). This protein is a citolisin, which belongs to the family of the actinoporins, purified from the sea anemone Stichodactyla heliantus. According to biological and structural studies of the native protein, the N-terminus region of St II is involved on the pore formation process in membranes. In order to elucidate the function and the importance of this region to the biological activity of St II, three analogs containing the first 30 residues of the N-terminus region of St II were synthesized and analyzed. The peptides were obtained by solid phase synthesis and altered through the substitution of a leucine or isoleucine for a tryptophan on the positions 2, 7 and 24. Circular dichroism (CD) and tryptophan fluorescence studies have been carried out to investigate conformational properties of the peptides. In order to evaluate the structural modification on aqueous solution, studies were performed in order to evaluate the pH, ionic strength and addition of the secondary structural-inducing solvent TFE. The interaction studies with micelles were performed using three different surfactants: sodium dodecil-sulfate (SDS), N-hexadecyl-N,N-dimethyl-3-ammoniumpropanesulfonate (HPS) and lysophosphatidylcholine (LPC), and using acrylamide as a fluorescence suppression agent. We have demonstrated that on solid phase peptides synthesis using the Fmoc/tBu strategy is a success. The purification process of peptides through HPLC, has also revealed to be efficient, once the material was obtained with high purity level above 95%. The experiments of hemolytic activity showed that the three peptides have activities in æM concentration, this results reinforce the idea that the 30 residues of the N-terminus region have an important role on pore formation in membranes. / Mestre

Diabetes mellitus is considered as one of world's most common metabolic diseases. Complicated treatment and increasing number of newly diagnosed patients, suffering from diabetes every year, shows the importance and necessity of research in this area. Some of the major aims of this research are the development of new therapeutically utilized drugs and defining the problems of insulin acting in human body. Insulin is a peptide hormone whose main physiological function is to regulate blood glucose level in organism connected with large impact on whole metabolism. Insulin acts through binding of its monomeric form to the insulin receptor. Upon binding to the receptor molecule of insulin undergoes specific structural changes, which put the hormone into an active state. As of now, the structure of the insulin's active monomeric form is still unknown. By testing binding affinities of many modified insulin analogues there was discovered strong evidence between structural conformation of the C-terminus of the B-chain and binding affinity to the receptor. The most crucial data, necessary for this work, were observed from the structure of highly active insulin analogues that possessed unique B26 turn, recently prepared and described by team of Dr. J. Jiráček, IOCB AS CR. The aim of this work was synthesis of...

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<p dir="ltr">A study on the impact of the position of the attachment of the photolabile, backbone amide linker, 4-formyl-3-hydroxy-5-nitrobenzoic acid, on the synthesis of a model cyclic pentapeptide was conducted. The peptide was synthesized on a solid support and cleaved photolytically. The crude product was analyzed for the effect of changing position by LC/MS, 1HNMR, and yield. The target peptide could not be identified convincingly by LC/MS or NMR. It was observed that attachment of the backbone amide linker to the N alpha of tyrosine provided the highest crude product yield.</p>

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The next generation of toxic chemicals and hazardous wastes from sophisticated chemical industries will demand the environmental agencies to employ biological methods over the conventional physical and chemical remediation methods. Over the past decade, natural metallo-enzymes have been identified to degrade some of the major chemical contaminants through electron transfer pathways. However, these natural enzymes are less stable in organic solvents and they are not effective for the degradation of toxic compounds such as polychlorinated biphenyls or dioxins. This thesis explores the use of protein design approaches to produce chemically and molecularly modified enzymes, which are highly stable, possess little substrate specificity, and have higher activity than the natural enzymes. The experiments presented in this thesis make use of solid phase synthesis and site-directed mutagenesis for the synthesis and production of these enzymes and popular chromatographic techniques for their purification. The partial characterization of these proteins revealed the essential structural features of these proteins, and their catalytic activity was demonstrated by the use of peroxidase assays. / Master of Science