Les cellules dendritiques (CDs) sont des cellules présentatrices d'antigènes agissant comme messagers entre l'immunité innée et adaptative. Elles sont activées par des molécules endogènes : Danger-Associated Molecular Patterns (DAMPS) ou exogènes : Pathogen-Associated Molecular Patterns (PAMPS). Ces molécules modulent l'expression des récepteurs à la surface des CDs, la production de cytokines et la libération d'exosomes dans le milieu extracellulaire. De plus, il est connu que le neuropeptide Vasoactive Intestinal Peptide (VIP) réduit l'activation des CDs par des PAMPS. Mon projet vise à étudier si l'activation des CDs par des DAMPS peut aussi être régulée par le VIP. Les résultats obtenus montrent que des DAMPs (ex : cristaux d'urate monosodique) et des PAMPS (ex : Lipopolysaccharide : LPS) provoquent une activation des CDs qui est diminuée en présence de VIP. Nous observons également que ce peptide diminue la production de cytokines et la quantité d'exosomes dans le milieu extracellulaire en réponse aux cristaux d'urate monosodique. Le VIP régule l'activation des CDs par des DAMPS ainsi que la production d'exosomes.

Les peptides de la famille du LAH4 sont des peptides cationiques capables de se replier en hélice α amphiphile. Ces peptides sont riches en histidines ce qui permet de moduler les interactions de ces peptides de manière pH dépendante et dans une gamme physiologique. Leurs capacités à interagir et perturber les membranes ont été utilisées pour divers applications biologique, et notamment pour l'amélioration de systèmes de transport de gènes.Le travail de cette thèse a été divisé en trois parties dans le but de caractériser de manière biophysique les différentes interactions ayant lieu lors de la livraison du système de transport de gènes à l’intérieur d’une cellule. L’interaction peptide-peptide : avec l’étude de l’agrégation en fibrilles de la VF1 ; l’interaction peptide-membrane : avec l’effet du LAH4L1 en présence de membranes ; et l’interaction peptide-ADN : avec le suivit de l’interaction entre le LAH4L1 et de l'ADN. / The LAH4 family consists of cationic amphiphilic peptides with propensity to fold in α-helical secondary structures. They contain histidines allowing the modulation of their interactions in a pH dependent manner in the physiological range. In membranes, at neutral or acidic pH the peptide assumes a transmembrane or an in planar configuration, respectively.In the field of gene delivery systems, peptides like LAH4 are used. They are able to firstly interact with different cargoes in order to form stable complexes, then interact with the cell membrane, and finally, promote to escape from the endosome.This PhD has been divided into three parts in order to characterize, with biophysical methods, the interactions occurring during the delivery of these gene systems: peptide-peptide interactions with a focus on the study of VF1 fibre formation; peptide-membrane interactions: with the investigation of the effect of LAH4L1 in different membranes; and peptide-DNA interactions, where the interactions of LAH4L1 with a small DNA fragment were measured.

Doctor of Philosophy / Department of Biochemistry / John M. Tomich / Peptide-based delivery systems show great potential as safer drug delivery vehicles. They overcome problems associated with lipid-based or viral delivery systems, vis-a-vis stability, specificity, inflammation, antigenicity, and tune-ability. We have designed and synthesized a set of 15 and 23-residue branched, amphiphilic peptides that mimic phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated spheres with 50-150 nm diameters (confirmed by TEM and DLS). Whereas weak hydrophobic forces drive and sustain lipid bilayer assemblies, these structures are further stabilized by β-sheet hydrogen bonding and are stable at very low concentrations and even in the presence of SDS, urea and trypsin as confirmed by circular dichroism spectroscopy. Given sufficient time, they fuse together to form larger assemblies and trap compounds of different sizes within the enclosed space. They are prepared using a protocol that is similar to preparing lipid vesicles. We have shown that different concentrations of the fluorescent dye, 5(6)-Carboxyfluorescein can be encapsulated in these assemblies and delivered into human lens epithelial cells and MCF-7 cells grown on coverslips. Besides fluorescent dyes, we have delivered the plasmid (EGFP-N3, 4.7kb) into N/N 1003A lens epithelial cells and observed expression of EGFP (in the presence and absence of a selection media). In the case of large molecules like DNA, these assemblies act as nanoparticles and offer some protection to DNA against certain nucleases. Linear peptides that lacked a branching point and other branched peptides with their sequences randomized did not show any of the lipid-like properties exhibited by the branched peptides. The peptides can be chemically decorated with target specific sequences for use as DDS for targeted delivery.

No description available.

Peptide recognition domains (PRD) bind to short linear motifs on their biological partners and are found in several cellular pathways including those found to be critical in tumorigenesis. In this study, I aimed to generate peptide probes against PRDs present on proteins involved in ovarian cancer. Using bioinformatics, I identified 66 potential PRDs present on these proteins. I then used peptide phage display to successfully generate peptides against 27 of the 66 domains. To validate my results, I performed an extensive literature review and structural analysis. For several cases, the phage-display derived binding preferences are similar to previously reported studies. However, for a subset of domains, I identified non-canonical binding preferences that have not been reported previously in literature. The binding preferences obtained in this study can be used to design intracellular probes for studying the role of these PRDs in biological pathways important in ovarian cancer.

Peptide recognition domains (PRD) bind to short linear motifs on their biological partners and are found in several cellular pathways including those found to be critical in tumorigenesis. In this study, I aimed to generate peptide probes against PRDs present on proteins involved in ovarian cancer. Using bioinformatics, I identified 66 potential PRDs present on these proteins. I then used peptide phage display to successfully generate peptides against 27 of the 66 domains. To validate my results, I performed an extensive literature review and structural analysis. For several cases, the phage-display derived binding preferences are similar to previously reported studies. However, for a subset of domains, I identified non-canonical binding preferences that have not been reported previously in literature. The binding preferences obtained in this study can be used to design intracellular probes for studying the role of these PRDs in biological pathways important in ovarian cancer.

Les travaux présentés dans ce mémoire ont été motivés par l'importance économique de l'élevage de la crevette bleue du pacifique Litopenaeus stylirostris dont les fortes mortalités sont principalement dues au développement de maladies bactériennes et virales. Ils ont consisté en la caractérisation des deux premiers membres d'une famille originale de peptides multifonctionnels présents chez les crevettes pénéides, les stylicines. Ces peptides, nommés stylicines 1 et 2, sont des peptides anioniques (pI < 6.0), formés d'une région amino-terminale riche en résidus de type proline et d'une région carboxy-terminale riche de treize résidus cystéines. Ces molécules sont synthétisées et stockées dans de petits granules présents dans le cytoplasme des hémocytes. Pour mieux appréhender leurs rôles dans la réponse immunitaire des crevettes à une infection par des Vibrio, leurs formes recombinantes ont été produites dans E. coli BL21 (DE3) plysS, purifiées et caractérisées. Les deux rstylicines présentent des activités antiproliférative et anticoagulante. Seule la rstylicine1 présente des activités antimicrobiennes : antifongique sur Fusarium oxysporum (CMI<2.5 µM), et antibactérienne (bactériostatique) sur Vibrio sp (CMI<80 µM). Ce peptide est également capable de se lier aux LPS des bactéries à Gram (-) (Kd= 9.6x10-8 M) et d'agglutiner V. penaeicida "in vitro". Enfin, l'existence de gènes codant des formes modifiées de la stylicine1, chez certaines crevettes, pourrait être en relation avec une diminution de la résistante des individus aux infections. / The work reported here was motivated by the economical importance of the pacific blue shrimp Litopenaeus stylirostris farming where high mortality rates are due to bacterial and viral diseases. It consists in the characterisation of two original peptides, the first members of a new multifunctional family of peptides from peneide shrimps, the stylicines. Those two peptides, named stylicines 1 and 2, are negatively charged (pI < 6.0), and characterised by a proline-rich N-terminal region and a C-terminal region containing 13 cysteine residues. Stylicines are synthesized by heamocytes where they are stored within small cytoplasmic granules. To understand the role of these peptides in the immune response of shrimps to a vibrio infection, their recombinant forms were produced in E. coli BL21 (DE3) plysS, purified and characterised. The two rstylicines display biological anti-proliferative and blood clotting activities. Only rstylicine 1 displays antimicrobial activities: antifungal against Fusarium oxysporum (MIC<2.5µM) and bacteriostatic against Gram (−) bacteria, Vibrio sp. (MIC<80µM). Moreover this peptide displays an LPS-binding activity (dissociation constant (Kd) of 9.6×10−8 M) and agglutinate Vibrio. penaeicida "in vitro". Finally, the presence of sequences coding for modified forms of stylicine 1 in some shrimp's genome may be in relation with their lower ability to survive infections.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Michael Kanost / Insects secrete antimicrobial peptides as part of the innate immune response. Most antimicrobial peptides from insects have antibacterial but not antifungal activity. We have characterized an antifungal peptide, diapausin-1 from hemolymph of a lepidopteran insect, Manduca sexta (tobacco hornworm). Diapausin-1 was isolated by size exclusion chromatography from hemolymph plasma of larvae that were previously injected with a yeast, Saccharomyces cerevisiae. Fractions containing activity against S. cerevisiae were analyzed by SDS-PAGE and MALDI-TOF MS/MS and found to contain a 45-residue peptide that was encoded by sequences identified in M. sexta transcriptome and genome databases. A cDNA for diapausin-1 was cloned from cDNA prepared from fat body RNA. Diapausin-1 is a member of the diapausin family of peptides, which includes members known to have antifungal activity. The M. sexta genome contains 14 genes with high similarity to diapausin-1, each with 6 conserved Cys residues. Diapausin-1 was produced as a recombinant protein in Escherichia coli. Purified recombinant diapausin-1 was active against S. cerevisiae, with IC₅₀ of 12 μM, but had no detectable activity against bacteria. Spores of some plant fungal pathogens treated with diapausin-1 had curled germination tubes or reduced and branched hyphal growth. Diapausin-1 mRNA level in fat body strongly increased after larvae were injected with yeast or with Micrococcus luteus. In addition, diapausin-1 mRNA levels increased in midgut and fat body at the wandering larval stage prior to pupation, suggesting developmental regulation of the gene. Our results indicate that synthesis of diapausin-1 is part of an antifungal innate immune response to infection in M. sexta. Biophysical analysis showed that diapausin-1 binds to the β-1,3 glucan component of the S. cerevisiae cell wall. A second insect peptide investigated in this project was M.sexta stress-response peptide 1(SRP1), an immune-related peptide upregulated under different stress conditions including immune-challenge. Preliminary results for NMR structure determination are presented. Most of the amino acid residue spin systems were assigned, and we determined the connectivities of many amino residues as a first step to solve the NMR structure. The circular dichroism spectrum of SRP1 indicates that the peptide lacks alpha-helical structure and may contain beta strands and turns.

Thesis advisor: Steven D. Bruner / Peptidic natural products are produced by diverse organisms ranging from bacteria to humans. These secondary metabolites can be assembled by the ribosome or by nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. The architectural complexity and biological activity of such compounds make them interesting targets for study. Frequently, nonribosomal peptides contain nonproteinogenic amino acid building blocks, and the biosynthetic routes to both ribosomal and nonribosomal peptides often utilize tailoring enzymes. These specialized enzymes catalyze mechanistically challenging reactions and provide peptidic natural products with structural motifs not normally found in proteins. Structural studies of these tailoring enzymes will further our understanding of biosynthetic pathways, and engineered tailoring enzymes could find use as promiscuous catalysts for the chemoenzymatic synthesis of natural product analogs. The L-tyrosine 2,3-aminomutase <italic>Sg</italic>TAM catalyzes the formation of &beta;-tyrosine from L-tyrosine, and is used in the biosynthetic pathway to the enediyne antitumor antibiotic C-1027. This enzyme contains the rare electrophilic prosthetic group 4-methylideneimidazole-5-one (MIO) and is homologous to the histidine ammonia lyase family of enzymes. While lyases form &alpha;,&beta;-unsaturated carboxylates as products, <italic>Sg</italic>TAM catalyzes additional chemical steps that result in an overall 2,3-amino shift. The precise mechanistic role of MIO in the ammonia lyase and aminomutase families of enzymes was actively debated for over 50 years. Here, we report the first x-ray crystal structure of an MIO-dependent aminomutase and detail the synthesis and characterization of mechanistic probes for this enzyme. Furthermore, we report several structures of <italic>Sg</italic>TAM bound to substrate analogs. These co-crystal structures reveal how <italic>Sg</italic>TAM achieves substrate recognition and suggest a specific role for MIO in catalysis. The results of our studies allow for the rational engineering of MIO-based aminomutases and ammonia lyases with altered physical properties and substrate specificities. Additionally, we are currently studying several enzymes involved in the biosynthesis of the tricyclic depsipeptide microviridin J. This ribosomal peptide natural product contains two lactones and one lactam, which are introduced by two enzymes belonging to the ATP-grasp ligase superfamily of proteins. Here, we detail the overexpression of these enzymes, MdnJ-B and MdnJ-C, in <italic>E. coli</italic> and report the optimization of conditions which lead to the crystallization of both enzymes. The structural characterization of MdnJ-B and MdnJ-C will lead to a greater understanding of macrocycle formation in ribosomal peptide biosynthesis, and engineered variants of these enzymes may find use as macrocylcization catalysts. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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No. of bitstreams: 2 Dissertação - Carolina Oliveira Matos - 2015.pdf: 5441476 bytes, checksum: 86d4c7bb5073b76109534a172cf579e5 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Antimicrobial peptides are part of the innate defense of several organisms, and represent candidate drugs in their natural form, allowing for the development of modified peptides with improved pharmacological profiles. In this context, this study aimed of the synthesis of Cn-AMP1, an antimicrobial peptide naturally isolated from green coconut water, which has activity against fungi, gram-positive and gram-negative bacterias well as effects on the proliferation of cancer cells and low toxicity to mammalian cells, the analogs [Trp9] Cn-AMP1 and [Gly9] Cn-AMP1 were also obtained by solid phase peptide synthesis using the Fmoc strategy. The characterization and purification of the peptides were performed by mass spectrometry and high-performance liquid chromatography. Structural studies of the peptides were performed by circular dichroism (CD) and nuclear magnetic resonance (NMR) in the presence of biomimetic enviroments. CD and NMR results indicated that the peptides do not present preferential conformations in aqueous solutions, however adopt helical conformations in membrane mimetic environments. CD studies have shown that Cn-AMP1 and [Gly9] Cn-AMP1 do not adopt show defined conformation in the presence of DPC micelles at different pH values, however the peptide [Trp9] Cn- AMP1 showed a small a-helical content in the presence of 100mM DPC. In the presence of SDS the spectra of all peptides showed helical profiles at both pH 4, pH 7 as well as in the absence of buffer. NMR experiments indicated the interaction of the peptides [Trp9] Cn-AMP1 and [Gly9] Cn-AMP1 with SDS micelles at pH 4 (25 °C), and structural calculations indicated that [Trp9]Cn- AMP1 adopts an α-helical conformation between Val2-Gly8, and that [Gly9] Cn- AMP1 adopts an α -helical conformation between Val2-Arg5. Dynamic light scattering and zeta potential experiments were performed to investigate the effect of addition of peptides into phospholipid vesicles. All of the peptides caused variations on the POPC:POPG (3:1) and POPC, LUVs hydrodynamic radios, however major changes were observed for the anionic vesicles due to the strong interaction between the arginine residue of the peptides and the negativelly charge of membrane. / Os peptídeos antimicrobianos fazem parte da defesa inata de vários organismos e representam candidatos a fármacos em sua forma natural, o que possibilita o desenvolvimento de peptídeos modificados com perfis farmacológicos melhorados. Neste contexto, o trabalho teve como objetivo a síntese do peptídeo antimicrobiano Cn-AMP1, originalmente isolado da água de coco verde, o qual apresenta atividade contra fungos, bactérias grampositivas e gram-negativas, efeitos sobre a proliferação de células cancerígenas e baixa toxicidade em células de mamíferos. Foram também sintetizados seus análogos [Trp9]Cn-AMP1 e [Gly9]Cn-AMP1 por síntese de peptídeos em fase sólida utilizando a estratégia Fmoc. A caracterização e purificação dos peptídeos foram realizadas por espectrometria de massas e cromatografia líquida de alta eficiência. Estudos estruturais dos peptídeos foram avaliados por Dicroísmo Circular (CD) e Ressonância Magnética Nuclear (RMN) na presença de meios biomiméticos. Os resultados de CD e RMN evidenciaram que os peptídeos se apresentam de forma desordenada em solução aquosa, porém adotam conformações helicoidais na presença de meios que mimetizam a membrana celular. Estudos detalhados de CD mostraram que os peptídeos Cn-AMP1 e [Gly9]Cn-AMP1 não apresentam estrutura definida em DPC a diferentes valores de pH, e o peptídeo [Trp9]Cn- AMP1 mostrou uma pequena estruturação em a-hélice na presença de 100mM de DPC. Em SDS todos os peptídeos apresentam um perfil de estrutura helicoidal em pH 4, pH 7 e sem a adição de tampão. Experimentos de RMN mostraram a interação dos peptídeos [Trp9]Cn-AMP1 e [Gly9]Cn-AMP1 com micelas de SDS em pH 4 a 25ºC. Cálculos estruturais demonstraram que o peptídeo [Trp9]Cn-AMP1 assume conformação em α-hélice entre os resíduos Val2-Gly8, ao passo que o peptídeo [Gly9]Cn-AMP1 assume conformação em α- hélice entre os resíduos Val2-Arg5. Experimentos de espalhamento de luz dinâmico e potencial zeta mostraram ainda o efeito da adição de peptídeos no tamanho e nas cargas superficiais de vesículas fosfolipídicas. Todos os peptídeos causaram variação no Dh de LUVs de POPC:POPG (3:1) e LUVs de POPC, tendo-se observado maiores alterações em vesículas predominantemente aniônicas de POPC:POPG, sendo favorecido pela maior aproximação e atração do resíduo de arginina presente nos peptídeos com a carga negativa extra das unidades de POPG.