Dissertations / Theses on the topic 'Peptide'

Part 1: Development of a racemisation free process of attaching the first amino acid to a solid resin support for peptide synthesis. To overcome the high degree of racemisation of the amino acid in this process, we have explored a new kind of method of attaching this first amino acid to a resin. This approach involves activating the alcohol of the polyamide resin, instead of activating the amino acid. To test this theory we used benzyl alcohol as a model for the resin, and activated the alcohol with diisopropylcarbodiimide to form an O-isourea derivative. A detailed kinetic study was carried out to establish the rate of reaction with different benzyl alcohols, which proved that solution phase formation of the benzyl esters was successful. We were unable to attach the first amino acid to the resin support, which we believe is due to the reagents being unable to interact with the alcohol sites which are inside the bead of the resin. Part 2: Development of a novel peptidomimetic constraint. Current interest in the development of β-turn stabilising constraints for biologically active peptide motifs led us to synthesise a novel peptidomimetic molecule which in its cis form would provide a scaffold to constrain the conformation of a linear peptide. In stereochemically pure form the BocNH and CO₂Et would be pointing in the right direction to mimic a β-bend, and could be a prototype for peptidomimetics based on e.g. cell adhesion and anti-aggregatory peptides. The synthesis proved demanding as this dioxopiperazine combines an α-diamino with a β-keto ester system, and although all linear precursors were satisfactorily synthesised, the final step which involved a cyclisation reaction did not work. To remove the complication of a β-keto ester system we then attempted to synthesise (36). (Fig. 3754) Again the pre-cyclisation stages were achieved satisfactorily to yield Z-NHCH(NHBoc)CONHCH(CH₂OH)CO₂CH₃

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.

La producció de proteïnes recombinants en plantes representa una oportunitat per a la seva obtenció i utilització comercial. L'objectiu principal d'aquesta tesi industrial ha estat el desenvolupament de sistemes vegetals de producció de proteïnes, eficients i competitius econòmicament, amb possibilitats de portar-les al mercat. Per fer-ho hem explorat dos sistemes: els cultius cel·lulars de Daucus carota i les fulles de Nicotiana benthamiana, cadascun d'ells amb els seus avantatges i limitacions. Com a prova de concepte, ambdós sistemes van ser utilitzats per a la producció d'"insulin-like growth factor 1" (IGF1), un pèptid d'alt valor afegit per a les indústries cosmètica i farmacèutica. S'assajaren vàries estratègies innovadores per a millorar els rendiments de producció augmentant l'expressió gènica i reduir costos de purificació del producte. A més, l'activitat biològica de l'IGF1 i els seus derivats produïts en planta va ser avaluada en comparació amb pèptids sintètics. Com a primera estratègia s'assajaren supressors del silenciament de l'ARN d'origen viral per tal d'incrementar l'expressió gènica. En assajos d'expressió transitòria amb la proteïna verda fluorescent com a marcadora, seleccionàrem la proteïna P1b del ipomovirus Cucumber vein yellowing virus (CVYV). Els nostres resultats amb línies cel·lulars de pastanaga sobreexpressores de l'IGF1 o el seu pèptid derivat CPP-IGF1 (variant dissenyada per a millorar la seva penetració en cèl·lules humanes) mostraren que en combinació amb P1b s'arribava a rendiments de producció 4 vegades majors que les línies sense el supressor del silenciament. A més, els pèptids foren dirigits al medi de cultiu per facilitar el seu aïllament mitjançant una simple clarificació. En assajos d'activitat, les fraccions obtingudes confirmaren ser capaces d'incrementar la divisió de fibroblasts humans. En relació amb l'estabilitat de la producció, s'observà una reducció propera al 33% després de vint-i-un cicles de propagació successius, per la qual cosa s'implementà la criopreservació de les línies transgèniques per mantenir els rendiments de producció originals, i així establir bancs cel·lulars per usos futurs. Alhora, es desenvolupà un sistema de producció transitòria de l'IGF1 i el CPP-IGF1 en fulles de N. benthamiana utilitzant un vector derivat del virus del mosaic del tabac, Tobacco mosaic virus (TMV). Aquest sistema va permetre reduir el temps d'obtenció del pèptid actiu, encara que en comparació amb la producció a les línies cel·lulars l'obtenció del producte no fou tan senzilla. Per tal de facilitar la purificació de l'IGF1 a partir de les matrius vegetals, aplicàrem una estratègia innovadora basada en fusions a oleosina per dirigir la producció a cossos lipídics. Aquesta tecnologia ja havia estat utilitzada en llavors, però no en cultius cel·lulars i escassament en fulles. Les nostres observacions mostraren la presència d'abundants cossos lipídics en nombrosos cultius cel·lulars incloent-hi els de D. carota amb l'excepció de les dues espècies model analitzades, Nicotiana tabacum i Arabidopsis thaliana. Desafortunadament, l'expressió estable de fusions a l'oleosina sembla que va afectar greument al creixement dels calls cel·lulars, pel que s'exploraren alternatives de la seva aplicació a la producció en fulles. Per tal d'augmentar la quantitat de cossos lipídics, la producció de les fusions a l'oleosina es realitzà simultàniament amb la d'inductors de l'acumulació de triacilglicerols utilitzant elements clau de la seva ruta biosintètica en A. thaliana: l'enzim DGAT1 i el factor de transcripció WRI1. Quan ambdós inductors foren co-expressats en combinació amb fusions a oleosina i l'IGF1 en plantes de N. benthamiana, es va obtenir fins 1 μg/g d'IGF1 unit als cossos lipídics, fàcilment aïllable i actiu. El nostre treball proporciona evidències que la utilització de supressors del silenciament de l'ARN, els vectors virals i la tecnologia de les oleosines contribueixen al potencial de les matrius vegetals per a la producció de proteïnes d'interès.
La producción de proteínas recombinantes en plantas representa una oportunidad para su obtención y uso comercial. El objetivo principal de esta tesis industrial ha sido el desarrollo de sistemas vegetales de producción de proteínas, eficientes y competitivos a nivel económico, con posibilidades de llevarlas al mercado. Para ello hemos explorado dos sistemas: los cultivos celulares de Daucus carota y las hojas de Nicotiana benthamiana, cada uno con sus ventajas y limitaciones. Como prueba de concepto, ambos sistemas fueron utilizados para la producción de “'insulin-like growth factor 1” (IGF1), un péptido de alto valor añadido para las industrias cosmética y farmacéutica. Se ensayaron varias estrategias innovadoras para mejorar los rendimientos de producción aumentando la expresión génica y para reducir costes de purificación del producto. Además, la actividad biológica de IGF1 y sus derivados producidos en plantas se evaluó en comparación con péptidos sintéticos. Como primera estrategia se ensayaron supresores del silenciamiento de ARN de origen viral para incrementar la expresión génica. En ensayos de expresión transitoria con la proteína verde fluorescente como marcadora, seleccionamos la proteína P1b del ipomovirus Cucumber vein yellowing virus (CVYV). Nuestros resultados con líneas celulares de zanahoria sobreexpresoras de IGF1 o su péptido derivado CPP-IGF1 (variante diseñada para mejorar su penetración en células humanas) mostraron que en combinación con P1b alcanzaban rendimientos de producción 4 veces mayores que las líneas sin el supresor del silencing. Además, los péptidos fueron dirigidos al medio de cultivo para facilitar su aislamiento por simple clarificación. En ensayos de actividad, las fracciones obtenidas confirmaron ser capaces de incrementar la división de fibroblastos humanos. En relación a la estabilidad de la producción, se observó una reducción cercana al 33% después de veintiún ciclos de propagación sucesivos, por lo que se implementó la criopreservación de las líneas transgénicas para mantener los rendimientos de producción originales, y así establecer bancos de líneas celulares para usos futuros. También se desarrolló un sistema de producción transitoria de IGF1 y CPP-IGF1 en hojas de N. benthamiana utilizando un vector derivado del virus del mosaico del tabaco, Tobacco mosaic virus (TMV). Este sistema permitió reducir el tiempo de obtención del péptido activo, aunque en comparación con la producción en líneas celulares la obtención del producto no fue tan sencilla. Con el fin de facilitar la purificación de IGF1 desde matrices vegetales, aplicamos una estrategia innovadora basada en fusiones a oleosina para dirigir la producción a cuerpos lipídicos. Esta tecnología ya había sido utilizada en semillas, pero no en cultivos celulares, y escasamente en hojas. Nuestras observaciones mostraron la presencia de abundantes cuerpos lipídicos en numerosos cultivos celulares, incluyendo los de D. carota, con la excepción de las dos especies modelo analizadas, Nicotiana tabacum y Arabidopsis thaliana. Desafortunadamente, la expresión estable de fusiones a oleosina pareció afectar gravemente el crecimiento de los callos celulares, por lo que se exploró la alternativa de su aplicación a la producción en hojas. Para aumentar la cantidad de cuerpos lipídicos, la producción de las fusiones a oleosina se realizó simultáneamente con inductores de la acumulación de triacilgliceroles, usando elementos clave de su ruta biosintética en A. thaliana: la enzima DGAT1 y el factor de transcripción WRI1. Cuando ambos inductores fueron co-expresados en combinación con fusiones de oleosina e IGF1 en plantas de N. benthamiana, se obtuvo hasta 1 μg/g de IGF1 unida a los cuerpos lipídicos, fácilmente aislable y activo. Nuestro trabajo proporciona evidencias de que la utilización de supresores del silenciamiento de ARN, los vectores virales y la tecnología de oleosinas contribuyen al potencial de las matrices vegetales para la producción de proteínas de interés.
The production of proteins in plant cell cultures and whole plants represents great opportunities to develop products for commercial use. The main objective of this industrial thesis was to develop economic and efficient plant production systems to bring proteins of interest to the market. We explored two different systems, Daucus carota cell cultures and Nicotiana benthamiana leaves, each having advantages and drawbacks depending on the intended use of the products. As a proof of concept, both systems were applied in the production of the human insulin-like growth factor 1 (IGF1), a high value peptide for the cosmetic and therapeutic industries. Innovative strategies to enhance gene expression and to facilitate product purification were used to improve yields and to reduce costs. Moreover, the biological activity of the produced IGF1 and derivatives was evaluated and compared to the chemically synthesized peptides to demonstrate the usefulness of production systems. Our first approach to enhance gene expression and improve peptide yields was with RNA silencing suppressors (RSSs). Using transient expression assays and the green fluorescent protein (GFP) as reporter, we selected the P1b from the Cucumber vein yellowing virus (CVYV) Ipomovirus as the RSSs to enhance gene expression in carrot cell cultures. Our results demonstrated that transgenic lines overexpressing IGF1 or the derivative CPP-IGF1 (a variant tailored to enhance the delivery to human cells) reached up to 4-fold higher peptide yields in combination with P1b than without. The IGF1 or CPP-IGF1 was targeted to the culture media being easily purified by simple clarification of suspensions. Moreover, we found that the media containing the produced IGF1 or CPP-IGF1 stimulated the division of human fibroblasts. A cryopreservation process was applied to the transgenic lines to avoid the reduction in peptide production found over successive propagation cycles. This allowed us to recover the original yields, opening up the possibility of establishing master cell banks. We also developed a transient production system of IGF1 and CPP-IGF1 using N. benthamiana leaves and a derived tobacco mosaic virus vector. This system resulted in similar yields of active peptides to cell cultures with the main advantage of shortening production times, although requiring more complex downstream purification. Our innovative strategy to facilitate the purification of IGF1 from plant matrices was the use of oleosin fusion technology for lipid droplet (LDs) targeting. This technology has been previously used in LD-rich seeds, but unexplored in plant cell cultures or LD-poor tissues such as leaves. Our work showed that model cell cultures from Nicotiana tabacum or Arabidopsis thaliana were an exception, as many other plant cell cultures, including D. carota cells, do contain a large number of LDs and are susceptible to produce oleosin fusion proteins. However, as the stable expression of oleosin fusions severely affected callus cell growth, we tested the technology in transient expression in leaves. Due to the low level of LDs in leaves, oleosin fusion proteins production was in combination with triacylglycerol (TAG) induction to increase LD content simultaneously. For this purpose, key components of the TAG biosynthetic pathway, A. thaliana derived elements such as the enzyme DGAT1 and the regulatory factor WRI1 were co-expressed with the IGF1 oleosin fusion proteins in N. benthamiana leaves. Using this strategy, we obtained yields up to 1 μg/g of IGF1 bound to LDs, easily purified and fully active. Our work provides evidence of the potential of plant matrices to produce valuable peptides. Also, the oleosin technology, the use of RSSs and viral vectors explored will serve to overcome some of the known limitations of plant systems to produce active products of industrial interest.

L’utilisation de peptides pour des applications biomédicales reste limitée, en raison de leur courte demi-vie biologique. Le greffage de polymères aux peptides a conduit à l’amélioration des propriétés du peptide. Les conjugués peptide-polymère peuvent être synthétisés par méthode convergente ou divergente. Alors que la première approche conduit à de faibles rendements, la deuxième est limitée à la polymérisation de monomères vinyliques. Nous proposons une méthode de synthèse divergente des conjugués, basée sur la fonctionnalisation des liaisons peptidiques, via le greffage de polyéthers. En effet, la polymérisation anionique par ouverture de cycle (AROP) des epoxides, à partir d’amorceurs amide, à été démontrée. Cette méthode polyvalente donne accès à des chaines de type PEG sans activation préalable. Les fonctions NH des amides de peptides ont été déprotonnées par une base phosphazène pour générer un amorceur de l’AROP. Des dipeptides cycliques (DKP) ont d’abord été utilisés pour démontrer la fonctionnalisation des fonctions NH. Des conditions de polymérisation ont été identifiées pour synthétiser de manière contrôlée des chaînes de polymères fonctionnalisées par une DKP. Le même système d’amorçage a été appliqué à un dipeptide, et des conjugués ont pu être synthétisés. Des essais ont été menés sur un tripeptide protégé et non protégé, mais se sont révélés peu concluants
Peptides as drugs are facing drawbacks such as short in-vivo half life and low resistance to enzymes, which limits a larger scale use. To overcome these shortcomings, conjugation of polymers to peptides leads to improvements of pharmacokinetic properties of the peptide. Peptide-polymers conjugates are synthesized either by convergent or divergent synthesis. While the first strategy faces low yields, the second one is limited to vinyl-based polymers. We aim to functionalize peptides on amide bonds by divergent synthesis of polyether from the peptide. Anionic Ring-Opening Polymerization (AROP) of epoxides has already proven to be feasible, from amide-based initiators. The approach is polyvalent and gives access to PEG-like polymers without activation of peptides prior synthesis. In this project, NH amide functions from small peptides were deprotonated by phosphazene base to generate an AROP initiator. First, cyclic dipeptides, were used to demonstrate the possible functionalization on NH functions. Polymerization conditions were identified for a controlled AROP, to afford polymers end-capped by a DKP. Initiator’s complexity was increased to protected linear dipeptide. Similar initiating system was used as previously, and conjugates could actually be synthesized. Initiating capacities of tri-peptides and protected tri-peptides were also investigated but were found to be inefficient

Les organismes vivants produisent des peptides antimicrobiens (PAMs) pour se protéger contre les microbes. La résistance croissante aux antibiotiques nécessite le développement de nouvelles stratégies thérapeutiques et les PAMs sont des candidats prometteurs pour résoudre ce problème. Ils possèdent une activité à large spectre et leur principal mécanisme d'action par perméation de la membrane engendre peu de phénomènes de résistance. Néanmoins, leur faible biodisponibilité empêche leur utilisation. Certaines limitations peuvent être surmontées en développant des mîmes de PAMs qui conservent leur activité mais avec un potentiel thérapeutique accru. Les peptoïdes (oligomères de N-alkylglycine) structurés en hélice cationique amphiphile sont de bons mimes de PAMs. Les peptoïdes sont plus flexibles que les peptides en raison de l'isomérie cis/trans des amides N,N-disubstitués ; cependant la conformation des amides peut être contrôlée par un choix judicieux des chaînes latérales. Le but de cette thèse est d'étudier l'influence de chaînes latérales(hydrophobes ou cationiques) bloquant la conformation des amides en cis et induisant une structure hélicoïdale de type PolyProline I (PPI) robuste, sur l’activité antibactérienne et la sélectivité de peptoïdes. La conception, la synthèse et l’étude conformationnelle de nouveaux oligomères peptoïdes cationiques portant des chaînes latérales de type tert-butyle et/ou triazolium ont été réalisées. Dans un premier temps, la synthèse en solution d'oligomères à base de tert-butyle a été développée puis une stratégie de synthèse en phase solide a été mise en place pour accéder aux oligomères à base de 1,2,3-triazolium. Ensuite, ces nouveaux oligomères ont été évalués pour leur activité vis à vis d’un panel de bactéries Gram-positive et Gram-négative, leur l'activité antibiofilm et leur sélectivité cellulaire. Enfin, pour visualiser les effets des peptoïdes amphiphiles sur les bactéries, une étude de microscopie a été réalisée
Living organisms produce antimicrobial peptides (AMPs) to protect themselves against microbes.The growing problem of antimicrobial resistance calls for new therapeutic strategies and the natural AMPs have shown ground-breaking potential to address that issue. They show broad-spectrum activity and their main mechanism of action by bacterial cell membrane disruption implies low emergence of resistance which makes them potent candidates for replacing conventional antibiotics. Nevertheless, few hurdles are impeding their use, notably poor bioavailability profile. Some of these limitations can be overcome by developing peptidomimetics of AMPs which exhibit antibacterial activities together with enhanced therapeutic potential. Peptoids (i.e. N-alkyl glycine oligomers) adopting cationic amphipathic helical structures are mostly competent AMP mimetics. From a conformational point of view, peptoids are fundamentally more flexible than peptides primarily due to the cis/trans isomerism of N,N-disubstituted amides but studies in this area have shown that cis amide conformation can be controlled by careful choice of side-chain to set a PolyProline I-type helical structure of peptoids. In this thesis, the genesis of novel amphipathic cationic peptoids carrying cis-directing tert-butyl and/or triazolium-type side-chains and their untapped potential to act against bacteria will be discussed comprehensively. First, the solutionphase synthesis of tert-butyl-based oligomers was developed. Second, novel method of solid-phase submonomer synthesis was optimised to access 1,2,3-triazolium-based oligomers. Then, the synthesised cationic oligomers were evaluated for their antibacterial potential, followed by antibiofilm activity and cell selectivity assays. In the end, to have insights on the mode of action of amphipathic peptoids, microscopy was carried out

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

A rich gallery of novel systems based on the sequence KLVFF (namely KLVFF, FFKLVFF, AAKLVFF, AAKLVAA, βAβAKLVFF, KLVFF-PEG, FFKLVFF-PEG, AAKLVFF-PEG, AAKLVAA-PEG, FF-PEG, FFFF-PEG) was synthesized and characterized. For the first time structure-properties relationships of this class of materials were systematically explored, with emphasis on self-association properties in both bulk and solution. Such a comparative investigation, essentially absent from the literature, provides insights into the underlying mechanism of amyloid fibrogenesis, given that KL VFF has been identified as critical for amyloid fibril formation of the amyloid-β peptide. In this respect, this virtually unique, bottom-up approach contributes to the development of therapeutics for Alzheimer's disease. Despite the fact that all the peptides were found to organize into predominantly β-sheet conformations (in a variety of solvents and in bulk) they exhibit a versatile range of morphological features, such as fibrils, twisted assemblies of protofilaments, hollow tubes and single tapes. Interestingly, FFKLVFF was found to exhibit strong fibrillization properties, while helically twisted ribbons were obtained from βAβAKL VFF. The self-organization of KLVFF towards fibrillar symmetries gives rise, under certain conditions, to time-dependant hydrogels with potential for application in tissue engineering. Notably, only FFKLVFF-PEG and FFFF-PEG retain the β-sheet conformation of the peptide precursor, and they form fibrils bearing a peptidic core surrounded by a corona of PEG chains. At high volume fractions packing of the FFKLVFF-PEG fibrils leads to the evolution of nematic and hexagonal columnar phases. Owing to their inherent biocompatible character, those hybrids carry great promise to substitute a number of conventional polymers in applications such as tissue engineering, cell growth and drug delivery. In the solid state, the interplay between two competing factors, polymer crystallization and peptide fibrillization, was studied for several conjugates. Two distinct cases were identified; solidification of FFKL VFF-PEG controlled by peptide- peptide interactions, while KLVFF -PEG and AAKL V AA-PEG crystals reflected the characteristics of PEG spherulites.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xviii, 169 p.; also includes graphics (some col.) Includes bibliographical references (p. 160-169). Available online via OhioLINK's ETD Center

A detailed description of the characteristics of antimicrobial peptides (AMPs) is highly demanded, since the resistance against traditional antibiotics is an emerging problem in medicine. They are part of the innate immune system in every organism, and they are very efficient in the protection against bacteria, viruses, fungi and even cancer cells. Their advantage is that their target is the cell membrane, in contrast to antibiotics which disturb the metabolism of the respective cell type. This allows AMPs to be more active and faster. The lack of an efficient therapy for some cancer types and the evolvement of resistance against existing antitumor agents make AMPs promising in cancer therapy besides being an alternative to traditional antibiotics. The aim of this work was the physical-chemical characterization of two fragments of LL-37, a human antimicrobial peptide from the cathelicidin family. The fragments LL-32 and LL-20 exhibited contrary behavior in biological experiments concerning their activity against bacterial cells, human cells and human cancer cells. LL-32 had even a higher activity than LL-37, while LL-20 had almost no effect. The interaction of the two fragments with model membranes was systematically studied in this work to understand their mode of action. Planar lipid films were mainly applied as model systems in combination with IR-spectroscopy and X-ray scattering methods. Circular Dichroism spectroscopy in bulk systems completed the results. In the first approach, the structure of the peptides was determined in aqueous solution and compared to the structure of the peptides at the air/water interface. In bulk, both peptides are in an unstructured conformation. Adsorbed and confined to at the air-water interface, the peptides differ drastically in their surface activity as well as in the secondary structure. While LL-32 transforms into an α-helix lying flat at the water surface, LL-20 stays partly unstructured. This is in good agreement with the high antimicrobial activity of LL-32. In the second approach, experiments with lipid monolayers as biomimetic models for the cell membrane were performed. It could be shown that the peptides fluidize condensed monolayers of negatively charged DPPG which can be related to the thinning of a bacterial cell membrane. An interaction of the peptides with zwitterionic PCs, as models for mammalian cells, was not clearly observed, even though LL-32 is haemolytic. In the third approach, the lipid monolayers were more adapted to the composition of human erythrocyte membranes by incorporating sphingomyelin (SM) into the PC monolayers. Physical-chemical properties of the lipid films were determined and the influence of the peptides on them was studied. It could be shown that the interaction of the more active LL-32 is strongly increased for heterogeneous lipid films containing both gel and fluid phases, while the interaction of LL-20 with the monolayers was unaffected. The results indicate an interaction of LL-32 with the membrane in a detergent-like way. Additionally, the modelling of the peptide interaction with cancer cells was performed by incorporating some negatively charged lipids into the PC/SM monolayers, but the increased charge had no effect on the interaction of LL-32. It was concluded, that the high anti-cancer activity of the peptide originates from the changed fluidity of cell membrane rather than from the increased surface charge. Furthermore, similarities to the physical-chemical properties of melittin, an AMP from the bee venom, were demonstrated.
Aufgrund der steigenden Resistenzen von Zellstämmen gegen traditionelle Therapeutika sind alternative medizinische Behandlungsmöglichkeiten für bakterielle Infektionen und Krebs stark gefragt. Antimikrobielle Peptide (AMPs) sind Bestandteil der unspezifischen Immunabwehr und kommen in jedem Organismus vor. AMPs lagern sich von außen an die Zellmembran an und zerstören ihre Integrität. Das macht sie effizient und vor allem schnell in der Wirkung gegen Bakterien, Viren, Pilzen und sogar Krebszellen. Das Ziel dieser Arbeit lag in der physikalisch-chemischen Charakterisierung zweier Peptidfragmente die unterschiedliche biologische Aktivität aufweisen. Die Peptide LL-32 und LL-20 waren Teile des humanen LL-37 aus der Kathelizidin-Familie. LL-32 wies eine stärke Aktivität als das Mutterpeptid auf, während LL-20 kaum aktiv gegen die verschiedenen Zelltypen war. In dieser Arbeit wurde die Wechselwirkung der Peptide mit Zellmembranen systematisch anhand von zweidimensionalen Modellmembranen in dieser Arbeit untersucht. Dafür wurden Filmwaagenmessungen mit IR-spektroskopischen und Röntgenstreumethoden gekoppelt. Circulardichroismus-Spektroskopie im Volumen komplementierte die Ergebnisse. In der ersten Näherung wurde die Struktur der Peptide in Lösung mit der Struktur an der Wasser/Luft-Grenzfläche verglichen. In wässriger Lösung sind beide Peptidfragmente unstrukturiert, nehmen jedoch eine α-helikale Sekundärstruktur an, wenn sie an die Wasser/Luft-Grenzfläche adsorbiert sind. Das biologisch unwirksamere LL-20 bleibt dabei teilweise ungeordnet. Das steht im Zusammenhang mit einer geringeren Grenzflächenaktivität des Peptids. In der Zweiten Näherung wurden Versuche mit Lipidmonoschichten als biomimetisches Modell für die Wechselwirkung mit der Zellmembran durchgeführt. Es konnte gezeigt werden, dass sich die Peptide fluidisierend auf negativ geladene Dipalmitylphosphatidylglycerol (DPPG) Monoschichten auswirken, was einer Membranverdünnung an Bakterienzellen entspricht. Eine Interaktion der Peptide mit zwitterionischem Phosphatidylcholin (PC), das als Modell für Säugetierzellen verwendet wurde, konnte nicht klar beobachtet werden, obwohl biologische Experimente das hämolytische Verhalten zumindest von LL-32 zeigten. In der dritten Näherung wurde das Membranmodell näher an die Membran von humanen Erythrozyten angepasst, indem gemischte Monoschichten aus Sphingomyelin (SM) und PC hergestellt wurden. Die physikalisch-chemischen Eigenschaften der Lipidfilme wurden zunächst ausgearbeitet und anschließend der Einfluss der Peptide untersucht. Es konnte anhand verschiedener Versuche gezeigt werden, dass die Wechselwirkung von LL-32 mit der Modellmembran verstärkt ist, wenn eine Koexistenz von fluiden und Gelphasen auftritt. Zusätzlich wurde die Wechselwirkung der Peptide mit der Membran von Krebszellen imitiert, indem ein geringer Anteil negativ geladener Lipide in die Monoschicht eingebaut wurde. Das hatte allerdings keinen nachweislichen Effekt, so dass geschlussfolgert werden konnte, dass die hohe Aktivität von LL-32 gegen Krebszellen ihren Grund in der veränderten Fluidität der Membran hat und nicht in der veränderten Oberflächenladung. Darüber hinaus wurden Ähnlichkeiten zu Melittin, einem AMP aus dem Bienengift, dargelegt. Die Ergebnisse dieser Arbeit sprechen für einen Detergenzien-artigen Wirkmechanismus des Peptids LL-32 an der Zellmembran.

Microgels are lightly cross-linked hydrogel particles in the sub-micrometer to micrometer size range with a capacity to drastically change their volume in response to changes in the external environment. Microgels have an ability to bind and store substances such as biomacromolecular drugs, notably proteins and peptides, and release them upon stimuli, making them potential candidates as drug delivery vehicles and functional biomaterials. This thesis aims at clarifying important factors affecting peptide-microgel interactions. These interactions were studied by micromanipulator-assisted light and fluorescence microscopy focusing on microgel deswelling in response to peptide binding, as well as re-swelling in response to peptide release or enzymatic degradation. To evaluate peptide uptake in microgels, solution depletion measurements were used whereas the peptide secondary structure was investigated by circular dichroism. In addition, the peptide and enzyme distribution within microgels was analyzed with confocal microscopy. Results presented in this thesis demonstrate that peptide incorporation into microgels, as well as peptide-induced microgel deswelling, increases with peptide length and charge density. In addition, results demonstrate that the peptide charge (length) rather than peptide charge density determines microgels deswelling. End-to-end cyclization is shown to not noticeably influence peptide-microgel interactions, suggesting that peptide cyclization can be used in combination with oppositely charged microgel carriers to improve the proteolytic and chemical stability of the peptide compared to the corresponding linear variant. Peptide secondary structure is found to drastically affect peptide incorporation into, and release from, oppositely charged microgels. Furthermore, it is shown that microgel charge density, peptide molecular weight, and enzyme concentration all greatly influence microgel bound peptide degradation. Of importance for applications, protective effects of microgels against proteolytic peptide degradation are observed only at sufficiently high microgel charge densities. Enzyme-mediated microgel degradation is shown to increase with increasing enzyme concentration, while an increased peptide loading in microgels causes a concentration-dependent decrease in microgel degradation. Taken together, results obtained in this work have provided some insight into factors of importance for rational use of microgels as delivery systems for protein or peptide drugs, but also in a host of other biomedical applications using weakly cross-linked polymer systems.

The glucagon-like peptide-1 receptor (GLP-1R) potentiates glucose-stimulated insulin release from pancreatic β cells and promotes correct β cell function, as such it is a validated target for the treatment of type 2 diabetes (T2D). GLP-1R is a Family B GPCR, activated by the cognate ligand GLP-1(7-36), a 30 residue peptide hormone secreted after eating, and Exendin4 (Ex4), a 39-residue synthetic peptide. Peptide ligands interact with both the large extracellular domain and core domain of GLP-1R. Core domain interaction is thought to activate the receptor. Whilst the interaction between the receptor extracellular domain and ligand is well characterised, the ligand-core domain interaction and subsequent activation is not fully understood. Herein, a combination of mutant peptides and non-peptide ligands based on a pyrimidine scaffold (Pm compounds) are used in HTR-FRET cAMP accumulation assays, using recombinant FlpIn-HEK293 cells expressing human GLP-1R, to characterise the activation profiles of these ligands to decipher the underlying activation mechanism at the GLP-1R core domain. Structure-function studies of Pm compounds showed a trifluoromethyl and sulphur dioxide group are essential for GLP-1R activation, and that they allosterically enhance GLP-1(9-36) and Ex4(9-39) cAMP signalling profiles independently from their own cAMP response. Insulin secretion assays showed Pm compounds potentiate insulin release from INS-1 832/13 cells in combination with truncated GLP-1(9-36), implicating the use of allosteric modulators as treatment for T2D. Truncated GLP-1(15-36) was capable of binding and activating GLP-1R with low affinity and low potency, yet analogously truncated Ex4(9-39) was an antagonist with high affinity. Previous studies had demonstrated GLP-1(15-36) was an antagonist, and peptide-mediated activity had been attributed to the amino-terminus. Furthermore, the Pm compound-mediated cAMP response at GLP-1R was potentiated by Ex4(9-39). Mutant peptide activation data suggest activating residues D15, V16 and S17 are situated more centrally within the peptide ligand, and an extension to the currently accepted GLP-1R activation model is proposed.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvii, 143 p.; also includes graphics. Includes bibliographical references (p. 106-111). Available online via OhioLINK's ETD Center

Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2004.
Includes bibliographical references (leaves 148-151). Also available in electronic version. Access restricted to campus users.

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CNPq
Peptídeos antimicrobianos são componentes importantes do sistema de defesa de diversos organismos contra possíveis invasores. Em geral, são pequenos (até 100 aminoácidos), catiônicos e anfipáticos. Eles têm despertado o interesse da comunidade científica por sua capacidade de atuação contra micróbios, que não conseguem desenvolver resistência a esses peptídeos. Ou seja, eles emergem como complemento e/ou alternativa ao uso dos antibióticos convencionais. Este trabalho desenvolveu um modelo computacional de um peptídeo híbrido de pediocina A (N-terminal) e plantaricina 149a (C-terminal), dois peptídeos bactericidas. Dados experimentais obtidos pelo grupo da prof. Dra. Rosângela Itri do IFUSP foram utilizados para modelagem e comparação dos resultados. Foram feitas simulações de MD do peptídeo interagindo com membranas puras e mistas de POPC e POPG utilizando os parâmetros do campo de força GROMOS 53A6 e 54A7. As simulações com uma unidade do peptídeo revelaram a atualização 54A7 era a mais adequado para modelagem desses sistemas. Os mapas de estrutura secundária mostraram que o peptídeo adquire configuração mais ordenada quando interage com membranas com maior quantidade de POPG em sua composição. As simulações com duas unidades do peptídeo sugeririam que o peptídeo interage e penetra na camada de POPG através da região Cterminal. Na simulação com membrana de POPC, nenhuma das porções terminais ficou estável no interior da membrana. O efeito do aumento da concentração de peptídeos foi examinado colocando cinco e dez unidades do peptídeo para interagir com as membranas. Na membrana de POPC, os peptídeos não formam um único aglomerado e causam pouca perturbação na bicamada. Já na membrana de POPG, o efeito da interação do aglomerado de peptídeos é acentuado, provocando grandes deformações na bicamada lipídica, praticamente a destruindo. Esse fenômeno sugere um possível mecanismo carpete para ação do peptídeo sobre a membrana fosfolipídica de bactérias.
Antimicrobial peptides are important components of defense system in various organizations against possible invaders. They are generally small (100 aminoacids), cationic and amphipathic. They have stimulated the interest of the scientific community for its ability to act against microbes that cannot develop resistance to these peptides. That is, they emerge as complement and/or alternative to the use of conventional antibiotics. This study developed a computational model of a hybrid peptide pediocin A (Nterminal) and plantaricin 149a (C-terminal), two bactericidal peptides. Experimental data obtained by the group of prof. Dr. Rosângela Itri (IFUSP) were used for modeling and compare the results. MD simulations were made of the peptide interacting with pure and mixed POPC and POPG membranes. These simulations were performed using the parameters of the force field GROMOS 53A6 and 54A7. Simulations with a single copy of the peptide revealed that the force field 54A7 was the most appropriate for modeling these systems. The secondary structure maps showed that the peptide acquires a more ordered configuration when interacting with membranes with higher amounts of POPG in its composition. The simulations with two copies of the peptide suggest that the peptide interacts and penetrates the POPG layer via the C-terminal part. In the simulation with POPC membrane, none of the end portions remained stable within the membrane. The effect of increasing the peptide concentration of was examined by placing five and ten copies of the peptide to interact with the membranes. In the POPC membrane, the peptides do not form a single cluster and they cause little disturbance in the bilayer. In the POPG membrane, the interaction of peptides cluster is enhanced, causing large deformation and practically destroying the lipid bilayer. This phenomenon suggests a possible carpet mechanism of action of the peptide on the phospholipid membrane of bacteria.

The protease thermolysin has been used directly to synthesise dipeptides from Fmoc-protected amino acids on a PEGA₁₉₀₀ solid support. The thermolysin-catalysed solid phase synthesis of longer peptides is reported. Fmoc-protected peptides as long as hexamers (poly-L-leucine) and tetramers (poly-L-phenylalanine and poly-L-tyrosine) were enzymatically synthesised. In each case enzymatic synthesis of peptides resulted in the formation of a library of peptides varying in length, the formation of which was caused by “scrambling” by the enzyme. Due to the reversible nature of the enzyme catalysed peptide bonds, it was believed that the aforementioned solid phase peptide libraries may be exploited to generate dynamic peptide libraries. From the Juliá-Colonna asymmetric epoxidation it is known that chalcone undergoes non-covalent interactions with the amino terminus of poly-L-leucine. Chalcone was therefore employed as a suitable template for the PEGA₁₉₀₀ immobilised poly-L-leucine libraries. However, it was found that the Fmoc-protecting group used in these libraries inhibited binding of chalcone to poly-L-leucine. As an alternative, unprotected poly-L-leucine libraries were generated in solution phase from dileucine using thermolysin. The catalytic activity of poly-L-leucine library members and poly-L-phenylalanine library members in the Juliá-Colonna epoxidation was investigated.  These investigations demonstrated that the peptide amplified by chalcone in the dynamic combinatorial libraries displayed an improved catalytic activity in comparison to other library members. This indicates that dynamic peptide libraries may be exploited as a tool for identifying potential catalysts for the Juliá-Colonna asymmetric epoxidation.

Plant natriuretic peptides (PNP) are novel peptides which, like in vertebrates, have been shown to have a function associated with water and salt homeostasis. Two PNP-encoding genes have been identified and isolated from Arabidopsis thaliana, namely; AtPNP-A and AtPNP-B. In this study, the focus was on PNP-B, which has not been extensively studied. Bioinformatic analysis was done on the AtPNP-B gene. This included the bioinformatic study of its primary structure, secondary structure, tertiary structure, transcription factor binding sites (TFBS) and its relation to other known proteins. The AtPNP-B gene was shown to be a 510 bp long, including a predicted 138 bp intron. AtPNP-B was also shown to have some sequence similarity with AtPNP-A and CjBAp12. The TFBS for AtPNP-B and OsJPNP-B were compared and they comprised of TFBS that are related to water homeostasis and pathogenesis. This suggested two possible functions; water stress and homeostasis and a pathogenesis related function for PNP-B. Following bioinformatic analysis, the heterologous expression of the AtPNP-B was attempted to investigate whether the AtPNP-B gene encoded a functional protein and to determine the functional role of PNP-B. However, expression was unsuccessful. An evolutionary study was then carried out which revealed that there were some plants without the intron such as, rice, leafy spurge, oilseed rape, onion, poplar, sugar cane, sunflower and tobacco. These plants would therefore be used for expression and functional studies in the future. The evolutionary studies also revealed that PNP-B had a relationship with expansins and the endoglucanase family 45. Other PNP-B related molecules were also obtained from other plant genomes and therefore used in the construction of a phylogenetic tree. The phylogenetic tree revealed that AtPNP-B clustered in the same group as CjBAp12 while AtPNP-A had its own cluster group. There were also other PNP-B like molecules that clustered in the same group as expansins (α- and β-). Thus, we postulate that, like PNP-A, PNP-B also has a possible function in water and salt homeostasis. However, due to the clustering iii of AtPNP-B into the same group as CjBAp12, a possible role of PNP-B in pathogenesis-related response is also postulated.

Abstract Natriuretic peptide hormones secreted from the heart are important in maintaining the volume and electrolyte balance and in regulation of blood pressure. The secretion of natriuretic peptides is stimulated by myocyte stretch and paracrine factors. However, the intracellular actions of these stimuli and the cellular and molecular mechanisms involved in the processing and secretion of natriuretic peptides are still largely unknown. In this study, a new model for studies of the natriuretic peptide system was developed using a novel natriuretic peptide from salmon. Salmon (Salmo salar) maintains its water and salt homeostasis despite the volume gain in fresh water and electrolyte gain in sea water. Thus, salmon is an ideal model to study the mechanisms regulating the extracellular volume and salt balance, like natriuretic peptides. Furthermore, comparative studies revealing the common characteristics in phylogenetically distinct species suggest the importance of these factors in the regulation of the natriuretic peptide system. A novel natriuretic peptide, salmon cardiac peptide (sCP), was cloned from salmon heart. Distribution of sCP was studied in a variety of vertebrates and its physiological effects were examined in in vitro and in vivo experiments in salmon and rats. The storage and release of sCP was studied using a salmon ventricle perfusion system and by analysing the molecular forms of stored and secreted forms. Factors modulating the secretion and circulating concentration of sCP, and cardiac peptide and sCP mRNA level in salmon were examined as well. The biosynthesis of sCP is strictly restricted to the heart. sCP is stored in myocytes in the prohormone form, while the secreted form is a 29-amino acid peptide in salmon. Mechanical load on isolated salmon ventricle and volume overload in intact salmon induced a rapid release of sCP. Exposure to hyperosmotic environment decreased the plasma sCP level. sCP increased diuresis and natriuresis, as well as relaxed preconstricted arteries from salmon and rats. Thus the storage, processing and release of sCP resembles those of mammalian ANP. The circulating level of sCP in salmon was markedly upregulated at increased temperatures. Upregulation resulted from decreased elimination rather than increased secretion of sCP, providing the first direct evidence that elimination is used for the regulation of the natriuretic peptide system. In conclusion, sCP is a promising model for studying the general factors regulating the cardiac natriuretic peptides.

Peptides derived from DNA-binding zinc finger proteins were synthesised with pairs of cysteine residues with i,i+7 and i,i+11 relative spacings introduced into their sequence. The sidechains of these cysteine residues were then alkylated with the well-known water soluble photochrome 3,3’-bis(sulfo)-4,4’-bis(chloroacetamino) azobenzene (BSBCA). The change of shape of the azobenzene dye in these peptide-dye conjugates allowed photocontrol of peptide structure and thus peptide-DNA interactions. For a single zinc finger helix, UV irradiation yielded a peptide conjugate with a dissociation constant with respect to its cognate DNA sequence of 100 nM with no binding apparent prior to irradiation. However, the relatively short half-life of BSBCA proved a stumbling block, particularly in the control of larger peptides using multiple azobenzenes to control several -helical structural elements within large peptides. In addition to the short half-life of cis-BSBCA under physiological conditions, multiple BSBCA switches attached to the same peptide were shown not to relax independently of each other. These results led to the design, synthesis and examination of novel photo switches sensitive to visible, rather than UV light, with improved light state half-lives and bidirectional optical switching. Initial studies on thioindigo-based switches proved that molecules sufficiently polar to be water soluble were inaccessible by concise synthetic routes. Attention was then turned to the synthesis of ortho-halogen substituted azobenzenes and investigation of several new conjugation strategies for linking these photosensitive molecules to peptides. Subsequent refinements to the design of the tetra-ortho-halogen substituted azobenzenes altered the position of UV/visible absorbance bands of the cis and trans isomers to create a 47 nm separation in the wavelengths of the n-π* absorbances of the isomers to allow effective bidirectional switching. These changes also improved the half-life of the cis state from 24 minutes at 20 0C to 3,256 minutes at 60 0C. One of these new azobenzenes was reacted with apoptosis-inducing Bak peptides with different cysteine spacings (i,i+7 and i,i+11). Less stringent control over the binding of these peptides to Bcl-xL was observed than with BSBCA, perhaps due to the more flexible nature of the new crosslinker, but the optical properties of this class of molecules suggest a little further development will yield much improved photoswitches.

Vaccination with immunogenic peptides offers a safe and specific way of inducing protection against pathogens, however as of yet there are no peptide-based vaccines available. The limitations on the therapeutic use of peptides are due to their poor immunogenicity and short life span in vivo. Peptide delivery systems act to circumvent these issues. The aims of this research were to investigate the ability of virus-like particles (VLP) from Rabbit haemmorhagic disease virus (RHDV) to deliver immunogenic peptides, to characterize the immune response to these particles, and to investigate whether baculovirus could also act as a delivery system. The vaccine peptides HAT (representing a T helper cell epitope) and HAB (representing the major B cell epitope) derived from the haemagglutinin antigen of influenza virus A/PR/8/34 were used as a model to investigate the ability of these virus particles to act as delivery vehicles to the immune system. A scheme for the production and purification of RHDV VLP was established. Expression of the capsid protein from RHDV in a serum-free recombinant baculovirus system using suspension cultures of up to 200 ml, and separation by isopycnic centrifugation on cesium chloride gradients led to high yields of purified RHDV VLP. Up to 20 mg of pure VLP could be obtained from an 800 ml culture of insect cells infected with recombinant baculovirus. In vitro testing revealed that RHDV VLP carrying the peptide HAT as a genetic fusion were processed by dendritic cells (DC), and that this peptide could be presented to induce activation of T cells. However, the purified RHDV VLP alone were not able to induce significant upregulation of cell activation markers CD40, CD86, and CD80. A preliminary in vivo study revealed that when RHDV VLP carrying the HAT peptide were delivered by an intraperitoneal injection in the absence of adjuvant, the immune response to the peptide was weak, therefore the route of delivery and the use of immune adjuvants with the VLP were optimised. Five different routes of delivery and two different immune adjuvants were compared. VLP were delivered through subcutaneous, intraperitoneal, transcutaneous, intramuscular and intranasal routes. Delivery of the VLP through each of these routes resulted in potent serum antibody responses. However, the strongest antibody responses were elicited when the VLP were delivered through the intraperitoneal or intranasal routes. Of these two routes, intranasal delivery gave the best mucosal responses at the lung surface, and was therefore chosen as the route of delivery for subsequent trials. CpG DNA and the wild-type baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) were tested as adjuvants for the RHDV VLP. These two adjuvants gave similar results, both acting to enhance a T[H]1 type response against the VLP, characterized by significantly increased levels of serum IgG2a and enhanced IFN-γ production. Two approaches were then tested: using the RHDV VLP as a peptide carrier with a CpG adjuvant, and using baculovirus particles directly as self-adjuvanting carriers for vaccine peptides. HAT and HAB peptides were chemically coupled to RHDV VLP. Mice that were vaccinated with these VLP mixed with a CpG adjuvant were able to raise low levels of specific antibody in the serum against influenza, and specific IgA against influenza was detected in the lung. These results indicated that, though the immune responses raised were modest, the RHDV VLP was able to deliver the vaccine peptides to the immune system. HAT and HAB peptides were chemically coupled to baculovirus particles. When mice were immunized with the baculovirus carrying the vaccine peptides, they raised significant levels of IgG1 (p<0.001) and IgG2a (p<0.05) against influenza in the serum, when compared to peptide delivered alone. A significant level of influenza-specific IgA was also detected in the lung at 10 ng/ml in the mice that received the baculovirus coupled with peptide. Analysis of splenocyte cytokines showed that these mice also responded to restimulation with IFN-γ production at around 100 pg/ml. This research revealed that RHDV VLP are able to act as carriers for vaccine peptides, however there are some limitations to their use with the HAT and HAB model peptides. It also showed that baculovirus can be rapidly modified to carry vaccine peptides by chemical conjugation, and that these peptides can be delivered to induce specific systemic and mucosal immunity, raising both B cell and cell mediated responses. Both virus particles have potential as components for new strategies for vaccination.

Thesis (Ph.D.)--Tufts University, 2000.
Submitted to the Dept. of Chemistry. Adviser: Clemens Richert. Includes bibliographical references (leaves 103-107). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Peptides are small proteins that are crucial in many biological pathways such as antimicrobial defense, hormone signaling, and virulence. They often exhibit tight specificity for their targets and therefore have great therapeutic potential. Many peptide-based therapeutics are currently available, and the demand for this type of drug is expected to continue to increase. In order to satisfy the growing demand for peptide-based therapeutics, new engineering approaches to generate novel peptides should be developed. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of peptides that have the potential to be effective scaffolds for in vivo peptide engineering projects. These natural RiPP peptides are enzymatically endowed with post-translational modifications (PTMs) that result in increased stability and greater target specificity. Many RiPPs, such as microcin J25 and micrococcin, can tolerate considerable amino acid sequence randomization while still being capable of receiving unique post-translational modifications. This thesis describes how we successfully engineered E. coli to produce the lasso peptide microcin J25 using a two-plasmid inducible expression system. In addition, we characterized the protein-protein interactions between PTM enzymes in the synthesis of micrococcin. The first step in micrococcin synthesis is the alteration of cysteines to thiazoles on the precursor peptide TclE. This step is accomplished by three proteins: TclI, TclJ, and TclN. We found that a 4-membered protein complex is formed consisting of TclI, TclJ, TclN, and TclE. Furthermore, the TclI protein functions as a central adaptor joining two other enzymes in the Tcl pathway with the substrate peptide.

Dermatophilosis commonly known as "lumpy wool" in sheep in Australia, is a skin disease of animals and man caused by Dermatophilus congolensis. In sheep dermatophilosis causes significant wool production losses both directly and indirectly due to down grading of wool and hides, low meat production and high mortality. particularly in young sheep. Control of the disease by vaccination is desirable but has only been partially successful and antigens are difficult to produce in sufficient quantity from D. congolensis. Dr T.M. Ellis and colleagues (Agriculture WA) have developed a vaccine that can produce a significant reduction in ovine dermatophilosis against several strains of the bacterium. In pen and field studies, the haemolysin based vaccine did not prevent initial infection but approximately 70% were protected against the development of lumpy wool. Their previous research showed that a vaccine prepared from a serine protease produced by D. congolensis also gave some protection against the disease. This antigen was expensive to prepare by conventional culture. An alternative approach is to use recombinant protein as an antigen. However, this is not a simple task if the protective antigens have not been identified and even if they have been, it can be difficult to prepare a recombinant protein in high yield. Random peptide libraries provide an alternative approach to the identification of peptides which might be useful in a vaccine. While these libraries have been used to identify epitopes and prepare diagnostic tests, their potential to produce antigens which could generate a protective immune response has still to be explored. The main aim of this thesis was to use random peptide libraries to identify new antigens which could be used in the future to immunise sheep and other animals against dermatophilosis. Because of the commercial availability of large random peptide libraries displayed on phage and flagellin there is an opportunity to produce low cost and immunologically potent peptide vaccines. To explore the effectiveness and reliability of random peptide libraries (Ph.D.™ and FliTrxTM libraries) for the selection of peptides, polyclonal antibodies against a recombinant serine protease from D. congolensis were used to pan the libraries. This recombinant serine protease was produced using a pQE expression vector and purified by immobilised metal affinity chromatography. Clones selected from the libraries were sequenced and the peptides aligned with the original amino sequence of serine protease. Many of the peptides aligned with varying homology to the serine protease sequence which demonstrated that the antibodies were selecting specific sequences from the libraries rather than just random peptides. To obtain peptides, which might be associated with a protective immune response to D. congolensis sheep which had been immunised with a crude enzyme preparation form D. congolensis by Dr T.M. Ellis were used as a source of antibodies. Four peptides from Ph.D.TM and three peptides from FliTrx™ libraries were chosen for vaccination of sheep. Sheep were given two doses of vaccine one month apart. Twentyone days after the second vaccination each sheep was challenged with a zoospore suspension of the MB and W14 strains of D. congolensis and the presence of lesions and their severity were measured at 7, 14 and 21 days after challenge. The immune response to D. congolensis antigens was also studied. There was a striking production of antibodies to antigens from D. congolensis induced by the peptides selected from the random peptide libraries. Vaccination with recombinant serine protease and with the peptides selected from the Ph.D.™ library increased the rate of resolution of the lesions caused by one strain of D. congolensis. The results in this thesis provide the first demonstration in large animals that phage displayed peptides can induce a specific immune response against an infectious agent.

Thesis advisor: Jianmin Gao
As the threat of microbial resistance to antibiotics grows, we must turn in new directions to find new drugs effective against resistant infections. Antimicrobial peptides (AMPs) and host-defense peptides (HDPs) are a class of natural products that have been well-studied towards this goal, though very few have found success clinically. However, as there is much known about the behavior of these peptides, work has been done to manipulate their sequences and structures in the search for more drug-like properties. Additionally, novel sequences and structures mimicking those seen in nature have been discovered and characterized. Herein, we demonstrate our ability to finely tune the antimicrobial activity of various peptides, such that they can be provided with more clinically desirable characteristics. Our results show that gramicidin A (gA) can be made to be less toxic via incorporation of unnatural cationic amino acids. This is achieved by synthesizing lysine analogues with diverse hydrophobic groups alkylated to the side-chain amine. Through exploring different groups, we achieved peptide structures with improved selectivity for bacterial over mammalian membranes. Additionally, we were able to achieve novel broad-spectrum gram-negative activity for gA peptides. In efforts to combat bacterial resistance to cationic antimicrobial peptides (CAMPs), we have directed our reported amine-targeting iminoboronate chemistry towards neutralizing Lys-PG in bacterial membranes. Originally incorporating 2-APBA into gA, we found this to hinder the peptide’s activity. However, we were successful in increasing the potency of gA3R, a cationic mutant of gA, towards S. aureus by using a co-treatment of this peptide with a Lys-PG binding structure. Currently, we are exploring this strategy further. Finally, we describe our work towards establishing a novel cyclic peptide library incorporating a 2-APBA warhead for iminoboronate formation with a given target. In this, we have achieved intermolecular reduction of iminoboronates, strengthening the stringency of library screening. Although we were unsuccessful in finding a potent hit for binding of the lipid II stem peptide, screening against human transferrin yielded selective hits. Currently we are investigating these hits to understand their activity and therapeutic potential
Thesis (PhD) — Boston College, 2017
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry

Investigations detailed in this thesis constitute a part of the continuing programme of research undertaken in our laboratory on the riboflavin carrier protein (RCP) with partic­ular reference to identification and synthesis of neutralizing antigenic determinants, design of relevant epitope mimetics with improved immunogenic characteristics and relationship between their secondary structures and immunological properties. The riboflavin carrier protein is elaborated as a reproductive stratagem to ensure ade­quate vitamin deposition in the developing oocyte in the chickens. The protein is scrupu­lously conserved through evolution in terms of physico chemical and immunological char­acteristics from fish through birds to mammals, including primates. In rodents and sub­human primates immunization with the heteroantigen viz., chicken egg white RCP leads to functional neutralization of the endogenous maternal protein resulting in curtailment of early pregnancy. Thus, the crucial role of RCP in maintenance of pregnancy is established and the protein identified as a potential candidate vaccine for immimocontraception. Fur­ther studies with the reduced and carboxymethylated (RCM) RCP as the immunogen re­veal that antibodies induced by RCM-RCP are equally effective in bioneutralization of the endogenous protein. So it can be surmised that the native folded structure of RCP is not obligatory for eliciting bioneutralizing antibodies. In an attempt to identify functionally relevant regions of the protein, a panel of monoclonal antibodies (MAbs) have been raised and characterized. One of the MAbs viz., 6J32Ci2 could bring about early fetal resorp-tion when injected to mice with confirmed pregnancies. These results prompted a detail molecular immunological approach to understand underlying mechanisms. The principal aims of the present investigations include: (1) identification of neutralizing epitopes; (2) synthesis of peptidyl sequences incorporating these determinants; (3) an understanding of the structure, antigenic and immunogenic characteristics of these peptides; (4) correlation of conformational and antigenic characteristics; (5) rational design and synthesis of peptide analogs with greater propensity to assume predicted secondary structures; (6) analysis of conformation dependency of peptide antigens and the importance of such conformation in generating an optimal B-cell response; (7) the efficacy of the antibodies elicited by these Peptide antigens in neutralizing endogenous protein with the ultimate aim of designing synthetic vaccines. Chapter 1 of this thesis deals with a general introduction summarizing the current status of knowledge regarding the chemistry and biology of RCP as well as synthetic pep­tides as potential immunogens. Chapter 2 outlines details of the experimental procedures adopted. Chapter 3 describes the results of investigations on the C-terminal fragment (residues 200-219) of cRCR The main consideration in selecting this sequence for the design of a potential peptide-based vaccine relied on the epitopic specificity of the neu­tralizing MAb 6S2C12. Epitope mapping using the Pepscan method revealed that the monoclonal antibody recognizes a core sequence corresponding to residues 203-210 of the cRCP. A 21-residue synthetic peptide (C-21) comprising this epitope was synthesized and antibodies elicited to the peptide conjugated to two different carriers, namely diphtheria toxoid and purified protein derivative (PPD) for T-cell help. In both active and passive immunoneutralization experiments, the peptide specific neutralizing antibodies interfered with the biological function of the protein and hence either protected from pregnancy or caused early fetal resorption in rodents as well as in sub-human primates. The conforma-tional properties of the peptide in aqueous buffers were analyzed from circular dichroism which revealed the absence of any ordered structure in the native C-21 peptide. Theoreti­cal predictions of secondary structure suggested a propensity for an t*-helical structure for this fragment in the native protein. Therefore, influence of the helix-promoting solvent, vizM 2,2,2,trifluroethanol (TFE) on the C-21 peptide was investigated. Addition of TFE resulted in spectral changes with negative bands at 208 and 222 nm and a positive band at 190 rim which are typical of an a-helix. To gain more information on the conformational characteristics of this peptide, it was considered worthwhile to stabilize the native peptide in an a-helical conformation based on simple rational design principles. Towards this end, four analogs of the parent peptide were synthesized and helix stabilization was sought to be achieved by introducing either salt bridges or back-bone conformational constraints such as by incorporating a-amino isobutyric acid at appropriate positions. In all the analogs, the core sequence, recognised by the neutralizing MAb 6B2C12 was maintained intact to ensure induction of antibodies capable of recognizing the native protein. CD spectral analysis of the analog peptides indicated that all the engineered peptides had varying degrees of enhanced helicities as compared to the parent peptide. The immunogenicity of each analog was studied by to the relevant peptide-diphtheria toxoid conjugates and analyzing their reactivities with the native protein by direct and competitive ELISA. The results revealed that these engi­neered conformational analogs axe highly immunogenic eliciting high titers of anti-protein antibodies. The relative affinities of these antibodies to bind cRCP were investigated. The antibodies to peptide analogs had higher affinities for the native protein and a positive correlation was found between the helical content of the peptide antigen in question and the relative affinity of corresponding antibody. The antibodies directed to all the peptide analogs could block the function of RCP resulting in early embryonic resorption when ad­ministered to pregnant mice. An interesting pattern of immunological cross-reactivity has been observed with the native and designed peptides. Antibodies raised to constrained helical analogs could bind the C-21 peptide which is structurally flexible. In contrast, the antibodies raised to the flexible native peptide antigen were inefficient in recognizing the structured peptides. The ability of all the peptide antibody to bind the native protein has been interpreted in terms of a conformationally flexible C-terminus region in cRCP. Chapter 4 details investigations on a 21-residue peptide (N- 21) from the N-terminiis (4-24) of the protein. Selection of this peptidyl sequence relied on theoretical prediction of potential sequential determinants on RCP other than at C-terminus as well as on the outcome of immunoneutralisation experiments using antibodies to egg yolk RCP which lacks the relevant C-terminal determinants. The structure of this peptide in solution was analyzed by two dimensional NMR and CD. NMR experiments revealed the presence of two structured regions in the peptide. Diagnostic nuclear Overhauser effects characteristics of reverse turns or short frayed helical segments over residues 3-9 and 18-21 of the peptide were obtained. CD spectra showed the presence of a strong, negative band at 204 nm over a wide range of solvent conditions, a feature which has been interpreted in terms of a "polyproline Il-like" segment encompassing residues 11-16 which corresponds to an interesting (X-Pro)^ repeat in the N-21 sequence. Specific antibodies were generated to this peptide as a conjugate with diphtheria tox­oid. Administration of the antipeptide antibodies could neutralize the protein in vivo as demonstrated by early embryonic loss in pregnant mice. In limited experiments the anti­peptide antibodies showed propensity to protect bonnet monkeys from pregnancy over a few consecutive ovulatory cycles when titres are maintained elevated by periodic boosting. To address the relationship between peptide structures and antigenicity, epitope mapping of this antipeptide antibodies as well- as the polyclonal antibodies to native RCP was undertaken using the Pepscan method. The results reveal that antigenic regions correspond well to conformationally well-defined elements of structure with the polyproline II-like seg­ment being a common antigenic determinant on both the peptide and the native protein. These observations are suggestive of the involvement of both the N and C-terminal regions of RCP in terms of its binding to putative plasma membrane receptors.