Dissertationen zum Thema „Biomimetické peptidy“

Práca sa zaoberá in silico dizajnom a validáciou peptidov založených na konotoxíne - MrIA, izolovaného z morských slimákov druhu Conus marmoreus a možnosti využitia týchto peptidov v liečbe neuroblastómu pomocou cielenia norepinefrínového transportéru. Päť peptidov založených na tomto konotoxíne bolo simulovaných pomocou simulácii molekulárnej dynamiky, ich trajektórie boli analyzované pre zistenie vlastností týchto peptidov. Dva homologické modely ľudského norepinefrínového transportéru boli vytvorené pre analýzu väzobných vlastností peptidov založených na konotoxíne ku norepinefrínovému transportéru. Peptidy boli následne syntetizované a použité na pokrytie apoferitínových nanočastíc s elipticínom uväzneným vnútri apoferitínu. Vytvorené peptidy a nanočastice boli ďalej skúmané pre objasnenie ich fyzikálo-chemických vlastností. Interakcie a cytotoxicita boli skúmané aplokáciou nanočastíc na bunky neuroblastómu a epitelu. Z in silico a in vitro analýz vyšiel YKL-6 peptid ako najlepší kandidát na ďalší výskum.

In this Master’s Thesis Research the results can be summarized from two major tasks: (1) In our first task, we utilized our two protein system (BAG-1 and HSP 70) as part of beta testing of a computational software 1 that can take three dimensional x-ray crystallography information about protein complexes and predict the strength of atom –atom interactions between amino-acid residues Open Contact predicts binding hotspots that can be used to identify short amino acid chains or peptides that mimic that particular binding segment of the larger protein. These peptides are called pepidyl-biomimetics. The peptide can potentially act as an antagonist drug by binding to the hotspot on protein A before protein B of the A-B complex can form. Two potential peptide candidates were identified. In particular, a helical peptide was discovered that demonstrated a variety of different types of atom-atom interactions. (2) Our second task is to experimentally test the helical peptide for its ability to block the binding that occurs between the 70-kilodalton Heat Shock Protein (HSP-70) and the Bcl-2 Associated Athanogene (BAG-1) Protein. As reviewed here, the binding between HSP-70 and BAG-1 elicits a cascade of cellular events that maintain high cancer growth rates and a greatly increased resistance to chemotherapy. In addition, BAG-1 has been implicated in a number of onco-signal pathways, as reviewed here, and its inhibition alone is believed to act as an agent against cancer cell growth

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 103-107). Also available in electronic version. Access restricted to campus users.

Le passage des médicaments a travers la membrane cellulaire représente souvent une limitation majeur dans un grand nombre de thérapies (anti-cancéreuse, anti-virale par exemple). Des peptides vecteurs connus comme les CPPs (cell penetrating peptides) ont été utilises avec succès pour introduire a l’intérieur des cellules diverses molécules (protéines, peptides, siRNA, quantum dots) et présentent un fort potentiel dans l'adressage de médicaments. Parmi les différents CPPs décrits dans la littérature la plupart sont des peptides basiques ou amphiphiles.Nous nous sommes intéressés a l'utilisation d’oligomères non charges construits a partir de motifs contraints mimes de dipeptides comme vecteurs de pénétration cellulaire. L'internalisation cellulaire et leur localisation ont été établies a l'aide de dérivés fluorescents par microscopie confocale. L' étude de pénétration cellulaire par mesure de fluorescence a montre que des oligomères de (3S)-amino-5-carbonylmethyl-2,3-dihydro-1,5-benzothiazepine-4(5H)-one] (DBT) sont aussi puissants que les oligomères d'arginine (oligoArg), vecteurs de référence. Par microscopie confocale nous avons montré que ces composés sont internalisés dans les lysosomes. L’efficacité d'internalisation de nos composés a été confirmé par une méthode de quantification par spectrométrie de masse MALDI-TOF développée dans notre groupe. Cette méthode repose sur l'utilisation conjointe d'un marqueur UV-absorbant dérivé de l'acide alfa-cyano-4-hydoxycinnamique (HCCA) et d'une matrice MALDI adaptée. Un effet important de discrimination spectrale est obtenu, permettant une amplification du signal de la molécule d' intérêt dans un mélange complexe. Ainsi les faibles concentrations internalisées peuvent être détectées. Grâce a cette technique et l'utilisation d'un étalon deutéré, nous avons calculé la concentration intracellulaire de deux CPP de référence l'octa-arginine et la pénétratine. Nous avons aussi étudier l’internalisation de petits oligomères construits a partir d'acide 2-aminomethyl-phenyl-acetique (AMPA). Par microscopie confocal nous avons constaté que ces petits oligomères sont internalisés par voie endo-lysosomale.L’efficacité de la pénétration cellulaire de ces petits oligomères aromatiques (oligoAMPA et oligoDBT) offre une nouvelle classe de vecteurs qui ont la particularité d’être non-cationiques et hydrophobes. De tels composés pourraient être utilisés pour la délivrance de médicaments dans le traitement des maladies comme le cancer, les maladies lysosomales ou la maladie d'Alzheimer. Afin de montrer que cette nouvelle classe de vecteurs est capable d'internaliser des composés biologiquement actifs, nous les avons associés a un inhibiteur puissant de la Cathepsine D (CD) la pepstatine. CD est une endopeptidase lysosomale qui dans des conditions normales est localisée dans les endosomes et les lysosomes. Pour certains cancers, la CD est surexprimée et secrétée a l’extérieur de la cellule. La CD est probablement impliquée dans la prolifération des cellules cancéreuses par l'activation de certains facteurs de croissances dans les endosomes. La pepstatine est une inhibiteur puissant de la CD. Cependant son efficacité thérapeutique potentielle est limitée par une faible capacité de pénétration des membranes cellulaires et une faible solubilité nécessitant de fortes doses pour l'inactivation de la CD in vitro et in vivo. Afin d’améliorer son efficacité et sa biodisponibilité, des conjugues de la pepstatine avec nos vecteurs de pénétration cellulaire, oligo (AMPA)4 et (DBT)4, et une partie solubilisante ont été développés. Certains de ces bioconjugués ont montre une toxicité élevée (IC50 = 2.10-6) in vitro sur différentes lignées cellulaires tumorales. Des tests in vivo sur des souris sont prévus pour le futur
As a part of a program for foldamer design two ¦Â-turn mimetics (3S)-amino-5-(carboxylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one or DBT and 2-aminomethyl-phenyl-acetic acid or AMPA were selected as frameworks from a molecular modeling study for their suitability to adopt helical structure. At first we developed a highly efficient scale up synthesis of the DBT moiety protected by 9-fluorenylmetoxycarbonyl (Fmoc) group. By standard solid phase peptide synthesis (SPPS) we synthesized DBT oligomers of different lenghts and modifications were introduced at their N-terminus. Our first task was to perform structural analysis of the oligomers by NMR and X-Ray. Numerous NOE interactions in the DBT pentamer and hexamer molecules were detected by NMR 2D NOESY experiments. These data strongly suggest the organization of these DBT oligomers. Small crystals were obtained from the same molecules in DMSO but at the time being their size is not importan t enough for X-Ray crystallography studies. In a parallel study we hypothesized that short oligomers constructed by DBT or AMPA frameworks could translocate the cellular membrane and could be used as new cell penetrating non-peptides - CPNP. Even though these compounds are not charged as most cell penetrating peptides (CPP)5 or CPNP, we considered that by virtue of their aromaticity, hydrophobicity and their well-organized structure they could have a non-specific interaction with the lipid bilayer and thus be internalized into the cell. Short oligomers were synthesized on Rink amide (RA) resin following SPPS methodology and labelled at their N-terminus with fluorescein isothiocyanate (FITC). At first the cellular uptake of the (DBT)2-4 oligomers in MDA-MB-231 breast cancer cells was analyzed by fluorescence emission measurement and compared to the potent and well-studied CPP octa-arginine (Arg)8 as a positive control and carboxyfluorescein as a negative control. The highest intracellular fluorescence intensity was found for (DBT)4 with a drastic decrease (>4-times) for (DBT)3 and (DBT)2 oligomers. Thus, the cellular uptake appeared length-dependent with an increase of the internalization with the oligomer size. Moreover, the amount of (DBT)4 that was internalized was more significant than that of (Arg)8 despite the fact that it is uncharged. By confocal microscopy we determined that (DBT)4 is mainly localized in the endosomes after 3 hours of incubation and in the lysosomes after 16 hours of incubation. Altogether, these data indicate the ability of these oligomers to target the endolysosomal pathway. Although most of the initial drug delivery studies aimed to avoid lysosomal addressing to prevent subsequent drug degradation, more recent studies demonstrated the relevant clinical utility to target this compartment for drug delivery in the treatment of lysosomal storage diseases, Alzheimer¡¯s disease, and cancer.While analyzing the internalization efficiency of our CPNP we decided to straightforward evaluate their concentration inside the cells. We studied our compounds internalization by total fluorescence emission measurement and by confocal microscopy but none of these techniques gave us the possibility to determine the exact amount of compound internalized per cell. A study reported by Burlina et al. brought a great improvement in proposing a highly reproducible quantification method based on MALDI-TOF MS to measure the concentration of the internalized peptides. However, after cell lysis, this method requires the capture of the biotin-labelled CPP by streptavidin coated magnetic beads. This step is particularly critical for the accuracy of the quantification. This is the reason why we decided to develop a new general methodology based on MALDI-TOF mass spectrometry (MS) which does not require any purification or separation steps. We studied the internalization of CPP/CPNP compou nds by using an UV light-absorbing tag alpha-cyano-4-hydroxycinnamic acid (HCCA) and preparing the samples in a neutral matrix such as alpha-cyano-4-hydroxycinnamic methyl ester (HCCE). This combination (HCCA tag and HCCE matrix) enabled us to discriminate MS signals induced by peptides of interest that were present in low concentration from those of unlabelled more abundant peptides. By addition of a precise amount of deuterated-HCCA-tagged CPP/CPNP prior the MALDI TOF MS experiment, the internalized CPP/CPNP could be quantified on the basis of the ratio between the [M+H]+ peaks of the deuterated and nondeuterated HCCA-tagged CPP.Another direction for research was to synthesize bioconjugates between our newly discovered CPNP and some biologically active compounds that are unable to cross the cell membrane. We selected pepstatine which is a powerful transition state inhibitor of the Cathepsin D (CD). Pepstatine while a very potent inhibitor of the CD is unable to cross the cellular membrane. Moreover pepstatine activity in vitro or in vivo is hampered by its poor solubility in water. CD is a soluble lysosomal aspartic endopeptidase synthesized in rough endoplasmic reticulum as preprocathepsin D (pCD).12 Upon entering the acidic endosomal and lysosomal compartments proteolytic cleavages of the pCD result in the formation of the active enzymatic form of CD. Under normal physiological conditions pCD is sorted to the lysosomes and found intracellularly but in some pathological and physiological conditions like cancer pCD/CD escape the normal targeting mechanism and is secreted from the cell. Once secreted to the outside, pCD can be endocytosed via M6PR or yet unknown receptor by both cancer cells and fibroblasts. The endocytosed pCD undergoes maturation into the enzymatically active CD. An enzymatic activity of CD outside of the cell or inside the endosomes could be responsible for the activation of several growth factors and growth factor receptors. Several groups have proven that the tumour growth is not inhibited by the powerful CD inhibitor pepstatine. These results exclude the importance of the CD enzymatic activity outside of the cell but as already mentioned pepstatine is unable to penetrate into the cell thus CD activation of growth factors inside the endosomes or the lysosomes is still a possibility. Different CPNP-Pepstatine conjugates were synthesized and tested in vitro for their ability to inhibit MDA-MB-231 breast cancer cells growth. Some of these conjugates showed high cytotoxicity, probably via a Cathepsin D inhibition in the endosomes or the lysosomes. One o f the most potent tested compounds was JMV4463. This compound was obtained by the conjugation of pepstatine with a CPNP as delivery system (AMPA4) and with solubilizing moiety composed of polyethylene glycol and D-Arginine residue. The good in vitro results obtained with the vectorized pepstatine encouraged us to perform in vivo tests. We performed scale up synthesis of JMV4463 in order to obtain enough product for anti-cancer activity on mice in the near future

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010.
Committee Chair: Bridgette Barry; Committee Member: David Collard; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Jake Soper; Committee Member: Mira Josowicz. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Dental enamel is an excellent example of a highly mineralised tissue, composed of hierarchically organised apatite mineral. This unique organisation gives enamel superior mechanical properties. However, when mature, enamel becomes acellular and unable to repair itself during traumatic or carious damage. The lack of self-repair requires dental intervention, where the common treatment of decayed enamel is to remove the affected and healthy tissue, and replace with restorative materials. The restorative materials, currently used, can cause further complications in the form of secondary caries or failure due to thermal and mechanical property mismatch with enamel. Problems associated with current restorative materials have driven researchers to explore biomimetic enamel treatment routes. To mimic the natural enamel formation, we can explore how proteins can guide mineral growth, in order to form enamel-like ordered mineral structures. In this thesis, the use of a synthetic, recombinant protein called an elastin-like protein (ELP) containing the analogue of the N-terminal of statherin (STNA15) was under investigation. Statherin is a protein present in saliva that is said to aid in the remineralisation of enamel. ELP with STNA15 (STNA15-ELP) has already shown promise in biomimetic mineralisation. This thesis investigated how conformation and structure of STNA15-ELP can be affected and manipulated by different chemical environments, surface constraint and crosslinking. The STNA15-ELP characteristics were related to formation of fluorapatite. STNA15-ELP conformation changed due to presence if salts in solution and whether or not it was constrained. We linked the conformational changes within STNA15-ELP, in solution versus on the surface, to two different routes of mineral formation. FAp formed in an uncontrolled manner with free STNA15-ELP. Ordered FAp formed via a precursor when STNA15-ELP was constrained on a surface. This work leads to an understanding of biomimetic mineralisation using STNA15-ELP. This information can aid in the design of novel biomimetic, enamel-like therapeutics.

Polyethylene glycol-stabilized lipodisks have emerged as a novel type of lipid-based nanoparticles with high potential as both drug carriers and biomimetic membranes. In this thesis we assess both of these applications, and show how the properties of the lipodisks can be further developed and optimized. Initially, we show that the antimicrobial peptides melittin, alamethicin and magainin 2, in spite of their very different physico-chemical properties and suggested modes of action on membranes, all have high affinity to lipodisks. Using melittin as a model peptide, we confirm a maintained antimicrobial effect of disk-formulated peptides. We also show that melittin dissociates slowly from the disks, resulting in extended drug release and prolonged antibacterial effect. Additionally, we present evidence that the peptide is protected against enzymatic degradation when formulated in the disks. Further, we develop a stable HPLC-MS system with immobilized lipodisks as model membranes. The stability of the system is confirmed by drug partitioning analysis using 15 different drug compounds. We also show how the lipodisk column can be supplemented with cyclooxygenase by in situ incorporation of the protein in the lipodisks. The specific binding of the protein to the disks is confirmed using QCM-D. Finally, by changing the polymer length and applying a new preparation protocol, we have optimized the lipodisks for use as drug carriers and biomimetic membranes. Previous lipodisk studies have been conducted on systems containing PEG-lipids with polymer molecular weights of 2000 or 5000 Da. Also, conventional protocols for the preparation of lipodisks typically require a PEG-lipid concentration of 15 mol% or more. Here we show that stable lipodisks can also be produced using PEG-lipids with a 1000 Da molecular weight polymer and that the use of shorter PEG-lipids dramatically improve the amount of lipodisks that can be immobilized on silica surfaces. Moreover, through the development of a method in which lipid mixtures are sonicated at low temperatures, we produce lipodisks containing as little as 2 mol% PEG-lipid. We present data verifying that these disks are superior to disks with higher PEG-lipid content in terms of their ability to incorporate externally added PEG-lipids functionalized with targeting agents.

Peptídeos antimicrobianos são vistos como alternativas promissoras a serem empregadas pela iindústria farmacêutica no controle de infecções causadas por microrganismos, como também na indústria alimentícia, onde podem desempenhar papéis como conservantes naturais de alimentos. Plantaricina149 é um membro deste grupo, sendo composto por 22 resíduos de aminoácidos, com natureza catiônica e atividade inibitória sobre algumas bactérias patogênicas. Neste trabalho, foram sintetizados diferentes peptídeos análogos à Plantaricina149 para investigar suas ações sobre microrganismos (bactérias e fungos), a fim de correlacionar estes estudos com a ação lítica do peptídeo em modelos de membrana diversos (monocamadas e vesículas fosfolipídicas). A interação de Plantaricina149 com estes sistemas foi monitorada pelas espectroscopias de dicroísmo circular e fluorescência, ensaios de tensão superficial, calorimetria e ressonância plasmônica de superfície, e mostrou ser altamente específica para superfícies fosfolipídicas que apresentam densidade de cargas negativas, tais como a membrana celular de bactérias. A interação eletrostática inicial que se estabelece entre o peptídeo e os fosfolipídios é de extrema importância, sendo capaz de induzir uma estruturação helicoidal na região C-terminal do peptídeo, enquanto a região Nterminal contribui com as interações hidrofóbicas necessárias para a penetração do peptídeo nas camadas fosfolipídicas levando a ruptura das mesmas. De forma semelhante, a atividade antimicrobiana de Plantaricina149a (e alguns de seus análogos) também mostrou ser resultado das interações das duas regiões da molécula, e foi afetada com a retirada ou modificação da região N-terminal do peptídeo. Com a deleção desta região, o peptídeo passou a ter somente ação bacteriostática sobre Staphylococcus aureus e Pseudomonas aeruginosa, perdendo a capacidade bactericida.
Antimicrobial peptides are seen as promising alternatives to be employed in pharmaceutical industry for controlling infections caused by microorganisms, and also in food industry, where they can play roles as natural food preservatives. Plantaricina149 is a member of this group, constituted of 22 amino acid residues, cationic in nature and presenting inhibitory activity against some pathogenic bacteria. In this work, different Plantaricina149 analog peptides were synthesized to investigate their action against microorganisms (bacteria and fungi), with the aim of correlating these studies with the lytic action of the peptide on several membrane models (phospholipid monolayers and vesicles). The Plantaricina149 interaction with these systems was monitored by circular dichroism and fluorescence spectroscopies, surface tension assays, calorimetry and surface plasmon resonance, and showed to be highly specific to phospholipid surfaces that present negative charge density, such as the bacteria cell membrane. The initial peptide-phospholipids electrostatic interaction is extremely important, and it is capable of inducing a helical structure in the peptide C-terminal region, while the Nterminal region contributes with the hydrophobic interactions needed to the peptide penetration in the phospholipid layers and to the disruption of them. Similarly, the Plantaricina149 antimicrobial activity has also proved to be a result of the interactions from the two regions of the molecule, and it was strongly affected by the removal or modification of the peptide N-terminal region. Promoting the deletion of this region has left the peptide only with a bacteriostatic action against Staphylococcus aureus and Pseudomonas aeruginosa, removing its bactericide ability.

Thesis (Ph. D.)--Materials Science and Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Sandhage, Kenneth; Committee Co-Chair: Kröger, Nils; Committee Co-Chair: Naik, Rajesh; Committee Member: Hud, Nicholas; Committee Member: Marder, Seth.

La diabetis és una malaltia que ha assolit proporcions epidèmiques. Aquest trastorn metabòlic es caracteritza per la hiperglucèmia crònica i està associat amb una elevada mortalitat cardiovascular i la reducció de l'esperança de vida. La diabetis mellitus tipus 1 (T1DM) està causada per la destrucció autoimmune de les cèl·lules beta pancreàtiques productores d'insulina. El reemplaçament de la massa de cèl·lules beta és un tractament prometedor per als pacients de T1DM, que té com a objectiu restaurar la normoglucèmia. El trasplantament d'illots pancreàtics ha demostrat el seu potencial com a teràpia de reemplaçament cel·lular, encara que la seva aplicació es troba limitada per diversos obstacles com l'escassetat d'illots pancreàtics. Per tant, es necessiten noves estratègies dirigides a generar una font abundant de cèl·lules productores d'insulina. Un enfocament prometedor és l'expansió de les cèl·lules beta adultes. No obstant, l'expansió in vitro de les cèl·lules beta implica la pèrdua del seu fenotip, probablement com a conseqüència de l'aïllament i la posterior dissociació dels illots en cèl·lules individuals. Aquests processos destrueixen el microambient cel·lular, alterant els contactes cèl·lula-cèl·lula i cèl·lula-matriu. L'enginyeria de teixits (ET) pot ajudar a superar aquests problemes creant teixits tridimensionals (3D), mitjançant l'ús combinat de cèl·lules i biomaterials que imitin la matriu extracel·lular (ECM) nativa. La hipòtesi de treball es va basar en el fet que la funcionalització de les matrius 3D amb motius de senyalització derivats de la ECM podria reconstituir el microambient dels illots, induint la rediferenciació de les cèl·lules expandides. La línia de cèl·lules beta de rata INS-1E, es va utilitzar com a prova de concepte per al model 3D funcionalitzat. El pèptid autoensamblable RAD16-I es va funcionalitzar amb els motius d'unió a integrina RGD, YIG, IKVAV, GEF i TWY. La caracterització de les matrius funcionalitzades va evidenciar que el pèptid RAD16-I mantenia la seva estructura característica de fulla beta en afegir els motius bioactius RGD, YIG i IKVAV. Per tant, les cèl·lules INS-1E es van encapsular amb els scaffolds biomimètics, els quals van promoure la supervivència cel·lular i la formació d'agregats cel·lulars, mimetitzant la conformació in vivo dels illots pancreàtics. A més, es va observar una major secreció d'insulina en les matrius funcionalitzades amb els pèptids YIG i IKVAV. Basant-nos en aquests resultats, es va intentar adaptar el model 3D per promoure la rediferenciació de les cèl·lules expandides dels illots humans. L'encapsulació de les cèl·lules desdiferenciades en la matriu de RAD16-I no va resultar ser un sistema adequat, per aquest motiu es van establir cultius en configuració sandvitx. Prèviament, es va induir la formació d'agregats cel·lulars (ICCs), propiciant així els contactes cèl·lula-cèl·lula. La conformació dels ICCs es va mantenir i va evolucionar en els cultius en sandvitx amb excel·lents valors de viabilitat. A més, els motius d'adhesió RGD i IKVAV van promoure la reexpressió dels marcadors de cèl·lula beta Ins, Pdx1, Nkx6.1 i MAFA. Aquests resultats indiquen que els cultius en configuració sandvitx, funcionalitzats amb RGD i IKVAV, són una plataforma 3D prometedora per induir la rediferenciació cap a un fenotip de cèl·lula beta, generant així cèl·lules que puguin ser utilitzades en la teràpia cel·lular de la diabetis.
La diabetes es una enfermedad que ha alcanzado proporciones epidémicas. Este trastorno metabólico se caracteriza por la hiperglucemia crónica y está asociado con una elevada mortalidad cardiovascular y la reducción de la esperanza de vida. La diabetes mellitus tipo 1 (T1DM) está causada por la destrucción autoinmune de las células beta pancreáticas productoras de insulina. El reemplazo de la masa de células beta es un tratamiento prometedor para los pacientes de T1DM, que tiene como objetivo restaurar la normoglucemia. El trasplante de islotes pancreáticos ha demostrado su potencial como terapia de reemplazo celular, aunque su aplicación se encuentra limitada por diversos obstáculos como la escasez de islotes pancreáticos. Por lo tanto, se necesitan nuevas estrategias dirigidas a generar una fuente abundante de células productoras de insulina. Un enfoque prometedor es la expansión de las células beta adultas. Sin embargo, la expansión in vitro de las células beta implica la pérdida de su fenotipo, probablemente como consecuencia del aislamiento y la posterior disociación de los islotes en células individuales. Dichos procesos destruyen el microambiente celular, alterando los contactos célula-célula y célula-matriz. La ingeniería de tejidos (TE) puede ayudar a superar estos problemas creando tejidos tridimensionales (3D), mediante el uso combinado de células y biomateriales que imitan la matriz extracelular (ECM) nativa. La hipótesis de trabajo se basó en que la funcionalización de las matrices 3D con motivos de señalización derivados de la ECM podría reconstituir el microambiente de los islotes, induciendo la rediferenciación de las células expandidas. La línea de células beta de rata INS-1E, se utilizó como prueba de concepto para el modelo 3D funcionalizado. El péptido autoensamblable RAD16-I se funcionalizó con los motivos de unión a integrina RGD, YIG, IKVAV, GEF y TWY. La caracterización de las matrices funcionalizadas evidenció que el péptido RAD16-I mantenía su estructura característica de hoja beta al añadir los motivos bioactivos RGD, YIG e IKVAV. Por lo tanto, las células INS-1E se encapsularon con los scaffolds biomiméticos, los cuales promovieron la supervivencia celular y la formación de agregados celulares, mimetizando la conformación in vivo de los islotes pancreáticos. Además, se observó una mayor secreción de insulina en las matrices funcionalizadas con los péptidos YIG e IKVAV. Basándonos en estos resultados, se intentó adaptar el modelo 3D para promover la rediferenciación de las células expandidas de los islotes humanos. La encapsulación de las células desdiferenciadas en la matriz de RAD16-I no resultó ser un sistema adecuado, por ese motivo se establecieron cultivos en configuración sándwich. Previamente, se indujo la formación de agregados celulares (ICCs), propiciando así los contactos célula-célula. La conformación de los ICCs se mantuvo y evolucionó en los cultivos en sándwich con excelentes valores de viabilidad. Además, los motivos de adhesión RGD e IKVAV promovieron la reexpresión de los marcadores de célula beta Ins, Pdx1, Nkx6.1 y MafA. Estos resultados indican que los cultivos en configuración sándwich funcionalizados con RGD e IKVAV, son una plataforma 3D prometedora para inducir la rediferenciación hacia un fenotipo de célula beta, generando así células que puedan ser utilizadas en la terapia celular de la diabetes.
La diabetes es una enfermedad que ha alcanzado proporciones epidémicas. Este trastorno metabólico se caracteriza por la hiperglucemia crónica y está asociado con una elevada mortalidad cardiovascular y la reducción de la esperanza de vida. La diabetes mellitus tipo 1 (T1DM) está causada por la destrucción autoinmune de las células beta pancreáticas productoras de insulina. El reemplazo de la masa de células beta es un tratamiento prometedor para los pacientes de T1DM, que tiene como objetivo restaurar la normoglucemia. El trasplante de islotes pancreáticos ha demostrado su potencial como terapia de reemplazo celular, aunque su aplicación se encuentra limitada por diversos obstáculos como la escasez de islotes pancreáticos. Por lo tanto, se necesitan nuevas estrategias dirigidas a generar una fuente abundante de células productoras de insulina. Un enfoque prometedor es la expansión de las células beta adultas. Sin embargo, la expansión in vitro de las células beta implica la pérdida de su fenotipo, probablemente como consecuencia del aislamiento y la posterior disociación de los islotes en células individuales. Dichos procesos destruyen el microambiente celular, alterando los contactos célula-célula y célula-matriz. La ingeniería de tejidos (TE) puede ayudar a superar estos problemas creando tejidos tridimensionales (3D), mediante el uso combinado de células y biomateriales que imitan la matriz extracelular (ECM) nativa. La hipótesis de trabajo se basó en que la funcionalización de las matrices 3D con motivos de señalización derivados de la ECM podría reconstituir el microambiente de los islotes, induciendo la rediferenciación de las células expandidas. La línea de células beta de rata INS-1E, se utilizó como prueba de concepto para el modelo 3D funcionalizado. El péptido autoensamblable RAD16-I se funcionalizó con los motivos de unión a integrina RGD, YIG, IKVAV, GEF y TWY. La caracterización de las matrices funcionalizadas evidenció que el péptido RAD16-I mantenía su estructura característica de hoja beta al añadir los motivos bioactivos RGD, YIG e IKVAV. Por lo tanto, las células INS-1E se encapsularon con los scaffolds biomiméticos, los cuales promovieron la supervivencia celular y la formación de agregados celulares, mimetizando la conformación in vivo de los islotes pancreáticos. Además, se observó una mayor secreción de insulina en las matrices funcionalizadas con los péptidos YIG e IKVAV. Basándonos en estos resultados, se intentó adaptar el modelo 3D para promover la rediferenciación de las células expandidas de los islotes humanos. La encapsulación de las células desdiferenciadas en la matriz de RAD16-I no resultó ser un sistema adecuado, por ese motivo se establecieron cultivos en configuración sándwich. Previamente, se indujo la formación de agregados celulares (ICCs), propiciando así los contactos célula-célula. La conformación de los ICCs se mantuvo y evolucionó en los cultivos en sándwich con excelentes valores de viabilidad. Además, los motivos de adhesión RGD e IKVAV promovieron la reexpresión de los marcadores de célula beta Ins, Pdx1, Nkx6.1 y MafA. Estos resultados indican que los cultivos en configuración sándwich funcionalizados con RGD e IKVAV, son una plataforma 3D prometedora para inducir la rediferenciación hacia un fenotipo de célula beta, generando así células que puedan ser utilizadas en la terapia celular de la diabetes.

Les métaux lourds, et en particulier les actinides, sont toxiques pour l'homme. La compréhension des mécanismes responsables de leur toxicité constitue un champ d'investigation important dans le domaine de la toxicologie. La compréhension des interactions de l’uranyle à l’échelle moléculaire est nécessaire pour prédire sa toxicité et pour concevoir des agents décorporants efficaces. Ce travail a pour objectif de contribuer à la caractérisation des sites d’interaction protéine-uranyle et à l’identification des facteurs clés gouvernant ces interactions. Pour obtenir des données thermodynamiques et structurales sur ces sites, deux stratégies ont été adaptées à l’étude des deux protéines humaines prédites comme cibles majeures de l’uranyle et dont les propriétés et structures sont très différentes. Les deux domaines structurés de la fétuine-A, ont été produits puis étudiés indépendamment par des méthodes physico-chimiques complémentaires incluant la spectroscopie RMN multidimensionelle afin d’obtenir des informations structurales sur les sites de liaison du métal dans la protéine. Afin d’élucider les interactions entre l’uranyle et l’ostéopontine, une protéine phosphorylée intrinsèquement désordonnée, nous avons conçu des peptides préorganisés en feuillet β comme modèles de sites de liaison de l’uranyle. Des acides aminés phosphorylés ont été introduits dans ces structures, permettant ainsi de reproduire l’environnement de coordination du métal dans la protéine. Les différences de structures et de propriétés entre biomolécules peuvent représenter un frein aux études d’affinité. Une sonde fluorescente non naturelle a donc été développée pour mettre au point une méthode de hiérarchisation des cibles de l’uranyle s’affranchissant de ces différences
Heavy metals, especially actinides, are toxic for humans. Understanding the mechanisms responsible for their toxicity is an important field of research in toxicology. Uranyl toxicity is still not well understood. The understanding of uranyl interactions at the molecular level is necessary to predict its chemical toxicity and to develop efficient chelating agents. This work aims at identifying uranyl binding sites in proteins and key factors that govern these interactions. To obtain thermodynamic and structural data, strategies were developed to study two proteins predicted as major uranyl targets which present different structures and properties. We took advantage of fetuin-A structure and studied the two structured domain of the protein by complementary physico-chemical methods including multidimensional NMR spectroscopy to acquire structural information on uranyl binding sites in this protein. In order to elucidate interactions between the metal and disordered phosphorylated proteins such as osteopontin, we designed peptides preorganized in β-sheet optimized to coordinate uranyl cation. We introduced amino acids containing phosphate groups and demonstrated that these peptides are relevant models to mimic uranyl binding sites found in phosphorylated proteins. Biomolecules display different structures and properties which may constitute an obstacle to affinity studies. A tool based on a non-natural fluorescent probe was developed to investigate and compare uranyl targets affinities

Cell adhesion to the extracellular matrix through cell-surface integrin receptors is essential to development, wound healing, and tissue remodeling and therefore represents a central theme in the design of bioactive surfaces that successfully interface with the body. This is especially significant in the areas of integrative implant coatings since adhesion triggers signals that regulate cell cycle progression and differentiation in multiple cellular systems. The interactions of osteoblasts with their surrounding extracellular matrix are essential for skeletal development and homeostasis and the maintenance of the mature osteoblastic phenotype. Our objective was to engineer integrin-specific bioactive surfaces that support osteoblastic differentiation and promote osseointegration by mimicking these interactions. We target two specific integrins essential to osteoblast differentiation the type I collagen receptor alpha2beta1 and the fibronectin receptor alpha5beta1. The central hypothesis of this project was that the controlled presentation of type I collagen and fibronectin binding domains onto well-defined substrates would result in integrin-specific bioadhesive surfaces that support osteoblastic differentiation, matrix mineralization, and osseointegration. We have demonstrated that these biomimetic peptides enhance bone formation and mechanical osseointegration on titanium implants in a rat tibia cortical bone model. We have also shown that the presentation of multiple integrin-binding ligands synergize to enhance intracellular signaling and proliferation. Finally, we demonstrate the advantage of the short biomimetic peptides over the native ECM proteins. This research is significant because it addresses current orthopaedic implant limitations by specifically targeting cellular responses that are critical to osteoblastic differentiation and bone formation. This biomolecular approach provides a versatile and robust strategy for developing bioactive surfaces that enhance bone repair and osseointegration of orthopaedic implants.

La biomimètica o biomimetisme són termes que simbolitzen el concepte “aprendre de la naturalesa”, és a dir, aprendre dels seus sistemes, processos i models, a fi d’utilitzar la natura com a font d’inspiració per solucionar problemes de l’home. El biomimetisme és actualment un concepte recurrent en l’àrea d’enginyeria de teixits i d’ell en sorgeixen idees per obtenir plataformes més elegants i sofisticades que puguin imitar millor les interacciones entre les cèl•lules i el seu ambient. Aquesta tesi pretén desenvolupar models, en dues i en tres dimensions, mitjançant la recreació d’un o més factors característics de l’ambient natural de la cèl•lula i que juguen un paper important en el comportament cel•lular. Se sap que tant les propietats químiques com les mecàniques de la matriu extracel•lular influeixen sobre les funcions cel•lulars. És per això que es va dissenyar un nou film polimèric que pogués combinar un hidrogel, amb propietats mecàniques variables, amb un monòmer reactiu capaç d’immobilitzar biomolècules. Degut a la complexitat del polímer dissenyat, va ser necessari recórrer a una tècnica de polimerització superficial molt versàtil com és la deposició química iniciada en fase vapor (més coneguda pel seu acrònim en anglès iCVD). Els polímers varen ser àmpliament caracteritzats i es va corroborar que podien ser modificats amb petites biomolècules com ara pèptids senyalitzadors. Les superfícies resultants són bioactives i permeten l’adhesió de cèl•lules endotelials. Unes altres superfícies biomimètiques, rellevants en l’àmbit de l’enginyeria de teixits d’os, es varen obtenir a partir d’una hidroxiapatita sintetitzada pel mètode de sol-gel submergint-la en diferents medis fisiològics. La dissolució i posterior reprecipitació dels ions proporcionen una capa d’apatita amb una composició similar a la que es troba in vivo. Els experiments evidencien la importància de partir d’un material relativament soluble. És per això que la hidroxiapatita pura no és capaç d’induir la precipitació d’aquesta apatita biomimètica in vitro. Diversos investigadors han relacionat la capacitat de formar apatita amb la bioactivitat del material, entenent bioactivitat com l’habilitat d’aquests materials de promoure la unió amb l’os. Per a l’enginyeria de teixits, però, és necessari un ambient tridimensional per tal de generar un teixit artificial. S’ha desenvolupat un nou model basat en l’ús d’un gel molt tou per tal d’obtenir un teixit dur com el de l’os. Malgrat que aquests dos conceptes poden semblar contradictoris, les cèl•lules adquireixen l’habilitat d’allargar-se ràpidament i crear una densa xarxa cel•lular dins d’aquest ambient poc restrictiu des d’un punt de vista mecànic. La consegüent contracció del sistema acaba formant un constructe més petit i resistent. Aquest és un sistema biomimètic ja que promou una gran interacció cel•lular i també la condensació de les cèl•lules, esdeveniments que tenen lloc també durant el desenvolupament de l’os i el cartílag. El model es va caracteritzar extensament amb cèl•lules ostoprogenitores MC3T3-E1 que es diferenciaren amb inducció química. A més a més, es va demostrar que l’ambient tridimensional podia promoure l’expressió espontània de marcadors osteogènics. Degut a les interessants propietats del sistema, el mateix model es va utilitzar per induir la diferenciació condrogènica de fibroblastos dermals humans. Aquests tipus cel•lular no ha estat gaire explorat en l’àmbit de l’enginyeria de teixits, malgrat que ofereix un gran potencial en teràpia regenerativa. Aquest treball proporciona proves de la capacitat condrogènica d’aquestes cèl•lules en el sistema tridimensional prèviament desenvolupat.
La biomimètica o biomimetismo son términos que simbolizan el concepto “aprender de la naturaleza”, es decir, aprender de sus sistemas, procesos y modelos, y utilizarlos como fuente de inspiración para solucionar problemas del hombre. El biomimetismo es actualmente un concepto recurrente en el área de ingeniería de tejidos y de este surgen ideas para obtener plataformas más elegantes y sofisticadas que puedan mimetizar mejor las interacciones entre las células y su ambiente. La presente tesis se centra en desarrollar modelos, tanto en dos como en tres dimensiones, mediante la recreación de uno o más factores que caracterizan el ambiente natural de la célula y que tienen su rol importante en el comportamiento celular. Se conoce que tanto las propiedades químicas como mecánicas de la matriz extracelular influyen en las funciones celulares. Debido a esto, se diseñó un nuevo film polimérico que pudiera combinar un hidrogel, con propiedades mecánicas variables, con un monómero reactivo, capaz de inmovilizar biomoléculas. Debido a la complejidad del polímero diseñado, fue necesario recurrir a una técnica de polimerización superficial muy versátil como es la deposición química iniciada en fase vapor (más conocida por su acrónimo en inglés iCVD). Los polímeros fueron ampliamente caracterizados y se corroboró que podían ser modificados con pequeñas biomoléculas como péptidos señalizadores. Las superficies resultantes son bioactivas y permiten la adhesión de células endoteliales. Se obtuvieron otro tipo de superficies biomiméticas relevantes en el ámbito de la ingeniería de tejidos de hueso, a partir de una hidroxiapatita sintetizada por el método sol-gel sumergiéndolas en diferentes medios fisiológicos. La disolución y posterior reprecipitación de los iones proporcionan una capa de apatita con una composición similar a la que se encuentra in vivo. Los experimentos evidencian la importancia de partir de un material relativamente soluble. Precisamente debido a esto la hidroxiapatita pura no es capaz de inducir la precipitación de esta apatita biomimética in vitro. Varios investigadores han relacionado la capacidad de formar apatita con la bioactividad del material, entendiendo bioactividad como la habilidad de estos materiales de promover la unión con el hueso. De todos modos, en ingeniería de tejidos, es necesario un ambiente tridimensional para generar un tejido artificial. Se ha desarrollado un nuevo modelo basado en el uso de un gel blando para obtener tejido duro como el del hueso. Aunque estos conceptos pueden parecer contradictorios, las células adquieren la habilidad de estirarse rápidamente y de formar una densa red celular dentro de este gel tan poco restrictivo desde un punto de vista mecánico. La consiguiente contracción del sistema acaba formando un constructo mucho más pequeño y resistente. Este es un sistema biomimético ya que promueve una gran interacción celular y también la condensación de las células, eventos que también ocurren durante el desarrollo de hueso y cartílago. El modelo se caracterizó extensamente con células osteoprogenitoras MC3T3-E1 que se diferenciaron bajo inducción química. Además, se demostró que el microambiente tridimensional podía promover la expresión espontánea de marcadores osteogénicos. Debido a las interesantes propiedades del sistema, el mismo modelo se usó para inducir la diferenciación condrogénica de fibroblastos dermales humanos. Este tipo celular no ha sido demasiado explorado en ingeniería de tejidos, a pesar de que puede tener un gran potencial en terapia regenerativa. Este trabajo proporciona pruebas de la capacidad condrogénica de estas células en el sistema tridimensional previamente desarrollado.
Biomimetics or biomimicry are terms that imply “learning from nature”, from its systems, processes and models, in order to use nature as inspiration to solve human problems. In tissue engineering, biomimetics is nowadays a recurrent term and a source of ideas to obtain more elegant and sophisticated platforms that could better mimic the interactions between cells and their environment. This thesis is focused on developing models both in two- and three-dimensions by recreation of one or more factors of the cell natural environment that are known to play an important role in cell behavior. Since both the chemical and mechanical properties of the extracellular matrix are known to effectively influence cell function, an innovative polymeric thin film was designed combining a hydrogel with tunable mechanical properties and a reactive molecule, capable to immobilize biomolecules. Due to the complexity of the polymers, a versatile technique such as initiated chemical vapor deposition (iCVD) was required for the synthesis. Extensive characterization revealed that nanostructured hydrogels were obtained and that small biomolecules, such as signaling peptides, could be attached on the surface. The final surfaces are bioactive and support endothelial cell attachment. Relevant biomimetic surfaces for bone tissue engineering could also be obtained from a sol-gel synthesized hydroxyapatite after immersion in different physiological media. The dissolution and posterior reprecipitation of the ions rendered a final apatite layer with a composition similar to that found in vivo. The experiments evidenced the importance of starting from a rather soluble material and, thus, pure hydroxyapatite was not able to promote apatite precipitation in vitro. This capacity has been related to the material bioactivity by many researchers in terms of its ability to bond to bone in tissue engineering applications. However, for tissue engineering a three-dimensional environment is required to build tissue-like constructs. A new model was developed based on the use of a very soft gel to obtain hard tissue. Although the concepts might seem to work in opposite directions, cells gain the ability to rapidly elongate and form a dense cellular network within this unrestrictive environment. Subsequent contraction of the whole system rendered a smaller and stronger final tissue-like construct. This system was considered biomimetic as it promotes high cell-cell interaction and cellular condensation, which are events that occur in bone and cartilage development. This system was extensively characterized with osteoprogenitor MC3T3-E1 cells that could undergo full osteogenic differentiation under chemical induction. More interestingly, the three-dimensional microenvironment was also able to promote by itself spontaneous expression of bone-related markers. Due to the interesting properties of this system, the same model was used to induce chondrogenic differentiation of human dermal fibroblasts. This cell type has been poorly explored for tissue engineering applications, but it might have great potential in future therapeutic platforms. This work provides proof of concept of chondrogenic potential of these cells in this three-dimensional system.

Dermicidina (DCD) é um gene mapeado no cromossomo 12, lócus 12q13.1, e codifica uma proteína de 110 aminoácidos, que sofre um processamento proteolítico, gerando peptídeos ativos. O peptídeo C-terminal (DCD-1L) de 48 aminoácidos tem uma carga -2, e exerce função antibacteriana e antifúngica, e o peptídeo C-terminal splice variante, denominado DCD-SV de 59 aminoácidos, tem carga neutra, e suas propriedades ainda não foram estabelecidas. Neste trabalho são apresentados os resultados da expressão, purificação e sequenciamento da DCD nativa produzida em E. coli BL21 transformada com o vetor pAE-DCD. Na segunda parte são descritas as análises físico-químicas e bioquímicas da interação dos peptídeos sintéticos DCD-1L e DCD-SV com vesículas lipídicas gigantes e vesículas unilamelar grandes sintetizadas com palmitoil-oleoil-fosfatidilcolina. As preferenciais estruturais dos peptídeos foram investigadas por espectroscopia de Dicroísmo Circular. Nossos resultados sugerem que a DCD-SV tem alta propensão para adotar uma estrutura helicoidal permitindo sua inserção e oligomerização em membranas biomiméticas, e possível formação de canais de condutância molecular.
Dermicidin (DCD) is mapped a gene on chromosome 12, locus 12q1.13 whose 110 amino acids protein is proteolytically processed to N and C-terminal peptides. The 48-amino acid C-terminal peptide (DCD-1L) has -2 net charges and display antibacterial and antifungal properties and the 59-amino acid splice variant C-terminal peptide (DCD-SV) has neutral net charge; however, its structure and biological function are unknown. Here we show the results of expression, purification and amino acid sequencing of recombinant DCD protein produced in E.coli transformed with pAE-DCD vector. We also describe the results of physical-chemical and biochemical analyses showing the visible differences between the interactions of DCD-1LL and DCD-SV synthetic peptides with giant unilamellar vesicles and large unilamellar vesciles made of palmitoyl-oleoyl phosphatidylcholine, used as biomimetic membranes. The structural preferences of peptides were analyzed by circular dichroism spectroscopy. Our results suggest that DCD-SV peptide has higher propensity to adopt helicoidal structure enabling it to insert into mimetic membranes, undergo oligomerization and formation of conductance channel.

Thesis (M. S.)--Materials Science and Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Tsukruk, Vladimir; Committee Member: Kalaitzidou, Kyriaki; Committee Member: Valeria Milam.

El cartílag articular té una capacitat limitada de creixement i regeneració i, els tractaments per restaurar la funció del teixit, després d’una lesió, són limitats i poc entesos per la comunitat mèdica. Existeix, per tant, un gran interès en trobar una solució pràctica i agradable pel pacient que aconsegueixi la reparació del cartílag. La enginyeria de teixits va sorgir per restablir teixits danyats usant noves plataformes terapèutiques basades en cèl·lules i/o biomaterials. Aquestes noves teràpies pretenen crear estructures similars al cartílag que imiten les propietats mecàniques i biològiques que trobem in vivo. En aquest context, l’ús de matrius biomimètiques que reprodueixin estructural i funcionalment el microambient natiu han despertat gran interès en aquest camp. Els pèptids auto-ensamblants representen candidats ideals per crear nínxols cel·lulars, ja que les seves nanofibres i propietats biomecàniques son similars a les de la matriu extracel·lular. En aquesta tesi, s’ha desenvolupat nous biomaterials sintètics amb gran potencial per la reparació de cartílag. Aquests estan basats en el pèptid auto-ensamblant RAD16-I decorat amb motius bioactius, amb l’objectiu de reproduir la matriu del cartílag. Donada la versatilitat del hidrogel RAD16-I, les noves matrius es van formar per simple mescla del pèptid RAD16-I amb molècules d’heparina, condroitin sulfat i decorina. Aquestes matrius bi-composades presenten bona estabilitat química i estructural a pH fisiològic i son capaces d’unir i alliberar, gradualment, factors de creixement. L’avaluació d’aquestes matrius es va dur a terme mitjançant dues estratègies in vitro diferents: la rediferenciació de condròcits articulars humans i la inducció del llinatge condrogènic en cèl·lules mare derivades de teixit adipós. Ambdós tipus cel·lulars son considerats una bona font cel·lular per obtenir constructes que reparin defectes al cartílag. Els resultats presentats en aquest treball mostren diferencies a nivell de comportament cel·lular, patrons d’expressió i propietats mecàniques entre els dos tipus cel·lulars i les diferents condicions de cultiu (matrius i medis). Cal destacar que els dos tipus cel·lulars es diferencien a un llinatge condrogènic en medi d’inducció i que els constructes presenten propietats mecàniques compatibles amb un sistema condrogènic. A més s’ha determinat que la presencia de molècules d’heparina a la matriu promou la supervivència de les cèl·lules mare derivades de teixit adipós. En conjunt, les noves matrius bi-composades representen un material fàcil de preparar i prometedor per promoure la diferenciació condrogènica. Finalment, part d’aquesta tesi s’ha centrat en el desenvolupament d’una nova matriu composta mitjançant la infiltració del pèptid RAD16-I amb cèl·lules en microfibres de policaprolactona (PCL). S’ha demostrat que aquesta nova combinació ofereix una estructura funcional i biomimètica, ja que proporciona suport mecànic per les fibres de PCL i a la vegada, facilita l’adhesió i el creixement cel·lular per l’hidrogel RAD16-I. El cultiu in vitro de condròcits humans desdiferenciats demostra que la nova matriu composada promou la supervivència cel·lular i el restabliment del llinatge condrogènic. En general, les propietats sinèrgiques de la nova matriu composada proporcionen una plataforma terapèutica ideal per ajudar a la reparació del cartílag.
El cartílago articular tiene una capacidad limitada de crecimiento y regeneración y, los tratamientos para restaurar la función del tejido, después de una lesión, son limitados y poco entendidos por la comunidad médica. Existe, por tanto, un gran interés en encontrar una solución práctica y agradable para el paciente que consiga la reparación del cartílago. La ingeniería de tejidos surgió para restaurar tejidos dañados usando nuevas plataformas terapéuticas basadas en células y/o biomateriales. Estas nuevas terapias pretenden crear estructuras similares al cartílago que imiten las propiedades mecánicas y biológicas que se dan in vivo. En este sentido, el uso de matrices biomiméticas que reproduzcan estructural y funcionalmente el microambiente nativo ha generado gran interés en este campo. Los péptidos auto-ensamblantes representan candidatos ideales para crear nichos celulares dado que, sus nanofibras y propiedades biomecánicas son similares a las de la matriz extracelular. En esta tesis, se han desarrollado nuevos biomateriales sintéticos con gran potencial para la reparación de cartílago. Éstos, están basados en el péptido auto-ensamblante RAD16-I decorado con motivos bioactivos, tratando de reproducir la matriz del cartílago. Dada la versatilidad del hidrogel RAD16-I, las nuevas matrices se formaron por simple mezcla del péptido RAD16-I con moléculas de heparina, condroitin sulfato y decorina. Estas matrices bi-compuestas presentan buena estabilidad química y estructural a pH fisiológico y son capaces de unir y liberar, gradualmente, factores de crecimiento. La evaluación de estas matrices se llevó a cabo mediante dos estrategias in vitro diferentes: la rediferenciación de condrocitos articulares humanos y, la inducción del linaje condrogénico en células madre derivadas de tejido adiposo. Ambos tipos celulares son considerados una buena fuente de células para obtener constructos que reparen defectos en el cartílago. Los resultados presentados en este trabajo muestran diferencias a nivel de comportamiento celular, patrones de expresión y propiedades mecánicas entre los dos tipos celulares y las diferentes condiciones de cultivo (matrices y medios). Cabe destacar que, ambos tipos celulares se diferencian a un linaje condrogénico en medio de inducción y que los constructos presentan propiedades mecánicas compatibles con un sistema condrogénico. Además, se ha determinado que la presencia de moléculas de heparina en la matriz promueve la supervivencia de las células madre derivadas de tejido adiposo. En conjunto, las nuevas matrices bi-compuestas representan un material fácil de preparar y prometedor para promover la diferenciación condrogénica. Por último, parte de esta tesis se ha centrado en el desarrollo de una nueva matriz compuesta mediante la infiltración del péptido RAD16-I con células en microfibras de policaprolactona (PCL). Se ha demostrado que esta nueva combinación ofrece una estructura funcional y biomimética, dado que, proporciona soporte mecánico por las fibras PCL y a su vez, facilita la adhesión y el crecimiento celular debido al hidrogel RAD16-I. El cultivo in vitro de condrocitos humanos desdiferenciados demuestra que la nueva matriz compuesta promueve la supervivencia celular y el restablecimiento del linaje condrogénico. En general, las propiedades sinérgicas de la nueva matriz compuesta proporcionan una plataforma terapéutica ideal para ayudar a la reparación del cartílago.
Adult articular cartilage has a limited capacity for growth and regeneration and, after injury, treatments to restore tissue function remain poorly understood by the medical community. Therefore, there is currently great interest in finding practical and patient-friendly strategies for cartilage repair. Tissue engineering has emerged to restore damaged tissue by using new cellular or biomaterial-based therapeutic platforms. These approaches aim to produce cartilage-like structures that reproduce the complex mechanical and biological properties found in vivo. To this end, the use of biomimetic scaffolds that recreate structurally and functionally the native cell microenvironment has become of increasing interest in the field. Self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural extracellular matrix (ECM). In the present thesis, new composite synthetic biomaterials were developed for cartilage tissue engineering (CTE). They were based on the non-instructive self-assembling peptide RAD16-I and decorated with bioactive motifs, aiming to emulate the native cartilage ECM. We employed a simple mixture of the self-assembling peptide RAD16-I with either heparin, chondroitin sulfate or decorin molecules, taking advantage of the versatility of RAD16-I. The bi-component scaffolds presented good structural and chemical stability at a physiological pH and the capacity to bind and gradually release growth factors. Then, these composite scaffolds were characterized using two different in vitro assessments: re-differentiation of human articular chondrocytes (ACs) and induction of human adipose derived stem cells (ADSCs) to a chondrogenic commitment. Both native chondrocytes and adult mesenchymal stem cells (MSCs), either bone marrow or adipose-tissue derived, are considered good cell sources for CTE applications. The results presented in this work revealed differences in cellular behavior, expression patterns and mechanical properties between cell types and culture conditions (scaffolds and media). Remarkably, both cell types underwent into chondrogenic commitment under inductive media conditions and 3D constructs presented mechanical properties compatible to a system undergoing chondrogenesis. Interestingly, as a consequence of the presence of heparin moieties in the scaffold cell survival of ADSCs was enhanced. Altogether, the new bi-component scaffolds represent a promising "easy to prepare" material for promoting chondrogenic differentiation. Finally, part of this thesis was focus on developing a composite scaffold by infiltrating a three-dimensional (3D) woven microfiber poly (ε-caprolactone) (PCL) scaffold with the RAD16-I self-assembling peptide and cells. This new combination resulted into a multi-scale functional and biomimetic tissue-engineered structure providing mechanical support by PCL scaffold and facilitating cell attachment and growth by RAD16-I hydrogel. The in vitro 3D culture of dedifferentiated human ACs evidenced that the new composite supports cell survival and promotes the reestablishment of the chondrogenic lineage commitment. Overall, the synergistic properties of the novel composite scaffold may provide an ideal therapeutic platform to assist cartilage repair.

Tissue engineers – in order to develop therapies for the treatment of complex neurological injuries and diseases – attempt to recreate elaborate developmental mechanisms in vitro. Neuronal precursor cells are excellent candidates for the study of developmental operations such as cell adhesion, differentiation, and axonal pathfinding. Hyaluronan (HA) is a common polysaccharide that is found extensively throughout the neuronal extracellular matrix (ECM), and can be functionalised and crosslinked to form stable hydrogels that support growing neuronal cells. Hyaluronan hydrogels can be modified chemically and mechanically to mimic the ECM of the developing brain, awarding control over mechanisms such as differentiation and axonal pathfinding. This thesis is concerned with the functionalisation and characterisation of HA hydrogels, ultimately in order to simulate vital properties of the developing brain. Here we show that HA hydrogels can be finely tuned mechanically (by modulating stiffness and viscosity), and chemically, by the conjugation of peptides that mimic the neural cell adhesion molecule (NCAM). NCAM mimics and novel mimics of sialylated NCAM significantly influence the differentiation of NSPCs in 2D and 3D. HA hydrogels successfully support long term culture of neural cells in 3D, and encourage the formation and extension of neurites of several cell types including human, mouse and rat neuronal precursor and stem cells. These results demonstrate for the first time that novel NCAM mimicking peptides can be conjugated to well defined hydrogel matrices that influence intricate developmental behaviours in 3D. Understanding how neural cells form functional networks is essential for the development of clinical approaches that attempt to address the injuries and diseases that affect these systems.

碩士
輔仁大學
化學系
96
The extracellular loop that might be the major part of odorant-binding domain in olfactory receptors. To mimic the extracellular loop binding site of olfactory receptor, a series of synthetic modified peptide with adding spacer Ala-Ala-Ala-Cys on N- and C- terminus of peptide are prepared with solid- phase synthesis protocol. The synthesis peptide are coated onto surface of piezoelectric electrode (PZ). The PZ biochip severe as a signal transducer of determine the binding affinity of modified synthetic peptides and odorants. Concept of molecular imprinting are introduced to enhance the selectivity of peptides. The result of these studies shoes the modified peptides would improve the selectivity between peptide and target odorant, the imprinted peptide also show higher sensitivity and specificity than non-imprinted peptide.

Acyl groups in biochemical reactions are activated as acyl adenylates; such intermediates are generated by a reaction with ATP. Acyl adenylates are mixed carboxylic-phosphoric anhydrides which are potentially useful as biomimetic reagents for acylation reactions in water. These species have been reported to be unstable and have been isolated without purification. Since the adenylate portion is necessarily complex because it originates from ATP, we reasoned that using a simple alkyl group in place of adenosine might allow the biomimetic process to proceed without the difficulties reported. Our laboratory has developed routes towards such acyl phosphate alkyl monoesters and we have used them for several applications. Such materials react rapidly and selectively with amines in order to produce amides. While reactions utilizing lanthanide ions allow for the selective monoacylation of diols through bis-dentate chelates of the lanthanide. However, the efficiency of diol acylation is limited due to significant hydrolysis of the phosphate reagent and the requirement of a stoichiometric amount of the lanthanide ion. Therefore, three distinct approaches towards improving the efficiency of lanthanide promoted acylation were investigated: addition of an inert co-solvent in an attempt to reduce hydrolysis, eliminating the stoichiometric requirement of the lanthanide by addition of MgII, and the development of immobilized lanthanides as catalysts for acylation. Finally, aminoacyl phosphates are biomimetically activated amino acids and in principle should function as peptide synthesis reagents. The stability and solubility of the activated materials in water presents an opportunity to perform aqueous peptide coupling; such a process is limited by the fact that common peptide coupling agents are either insoluble or unstable in water. Therefore, we investigated the reactions of aminoacyl phosphates with amino acid esters. We find that peptides form readily in buffered solutions, establishing a basis for a general protocol for aqueous amino acid coupling and could be adapted for applications such as solid phase peptide synthesis.

abstract: [FeFe]-hydrogenases are enzymes for the reduction of protons to hydrogen. They rely on only the earth abundant first-row transition metal iron at their active site (H cluster). In recent years, a multitude of diiron mimics of hydrogenases have been synthesized, but none of them catalyzes hydrogen production with the same exquisite combination of high turnover frequency and low activation energy as the enzymes. Generally, model complexes fail to include one or both of two features essential to the natural enzyme: an intricate array of outer coordination sphere contacts that constrain the coordination geometry to attain a catalytically optimal conformation, and the redox non-innocence of accessory [FeS] clusters found at or near the hydrogen-activating site. The work presented herein describes the synthesis and electrocatalytic characterization of iron-dithiolate models designed to incorporate these features. First, synthetic strategies are developed for constructing peptides with artificial metal-binding motifs, such as 1,3-dithiolate and phosphines, which are utilized to append diiron-polycarbonyl clusters onto a peptide. The phosphine-functionalized peptides are shown to be better electrocatalysts for proton reduction in water/acetonitrile mixtures than in neat acetonitrile. Second, we report the impact of redox non-innocent ligands on the electrocatalytic properties of two types of [FeFe]-hydrogenase models: dinuclear and mononuclear iron complexes. The bidentate, redox non-innocent α-diimine ligands (N-N), 2,2'-bipyridine and 2,2' bipyrimidine, are used to create complexes with the general formula (μ-SRS)Fe2(CO)4(N-N), new members of the well known family of asymmetric diiron carbonyls. While the 2,2'-bipyridine derivatives can act as electrocatalysts for proton reduction, surprisingly, the 2,2'-bipyrimidine analogues are found to be inactive towards catalysis. Electrochemical investigation of two related Fe(II) complexes, (bdt)Fe(CO)P2 for bdt = benzene-1,2-dithiolate and P2 = 1,1'-diphenylphosphinoferrocene or methyl-2-{bis(diphenylphosphinomethylamino}acetate, related to the distal iron in [FeFe]-hydrogenase show that these complexes catalyze the reduction of protons under mild conditions. However, their reactivities toward the external ligand CO are distinguished by gross geometrical differences.
Dissertation/Thesis
Ph.D. Chemistry 2013

Biomimetics are a growing field with several applications in Bioengineering. This work focuses on two examples of biomimetic systems applied to the fields of Biocatalysis and Biosensing. Enzymes are highly versatile catalysts present in biological systems with a coveted technological potential. Enzymes tend to be large and complex proteins and the development of simpler Biomimetic catalysts has been a long-term goal. In this work, two computationally designed protein-based peptides, RD01v2 and RD02, mimicking metalloprotease activity, were synthesized by SPPS and purified by Preparative HPLC Chromatography. The characterization process was divided in folding studies by far-UV CD spectroscopy and hydrolytic activity studies. All the assays were accomplished for a range of pH from 7 to 10, with and without added Zinc (II) at constant temperature. A Zinc titration at all these pH values, with the mentioned conditions, was performed to calculate the apparent dissociation constant (KpepZn,App in the range of 105 M-1). RD peptides presented low catalytic hydrolytic activity towards 4-nPA with second order rate constant (k2 <1 M-1s-1). The second exampled studied included the development of stimuli responsive materials mimicking the sense of olfaction.

Thesis (Ph. D.)--University of Illinois at Urbana-Champaign, 2007.
Source: Dissertation Abstracts International, Volume: 68-06, Section: B, page: 3796. Adviser: Mary S. Gin. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

Indiana University-Purdue University Indianapolis (IUPUI)
Hydrogels prepared from photopolymerization have been used for a variety of tissue engineering and controlled release applications. Polymeric biomaterials with high cytocompatibility, versatile degradation behaviors, and diverse material properties are particularly useful in studying cell fate processes. In recent years, step-growth thiol-ene photochemistry has been utilized to form cytocompatible hydrogels for tissue engineering applications. This radical-mediated gelation scheme utilizes norbornene functionalized multi-arm poly(ethylene glycol) (PEGNB) as the macromer and di-thiol containing molecules as the crosslinkers to form chemically crosslinked hydrogels. While the gelation mechanism was well-described in the literature, the network properties and degradation behaviors of these hydrogels have not been fully characterized. In addition, existing thiol-ene photopolymerizations often used type I photoinitiators in conjunction with an ultraviolet (UV) light source to initiate gelation. The use of cleavage type initiators and UV light often raises biosafety concerns. The first objective of this thesis was to understand the gelation and degradation properties of thiol-ene hydrogels. In this regard, two types of step-growth hydrogels were compared, namely thiol-ene hydrogels and Michael-type addition hydrogels. Between these two step-growth gel systems, it was found that thiol-ene click reactions formed hydrogels with higher crosslinking efficiency. However, thiol-ene hydrogels still contained significant network non-ideality, demonstrated by a high dependency of hydrogel swelling on macromer contents. In addition, the presence of ester bonds within the PEGNB macromer rendered thiol-ene hydrogels hydrolytically degradable. Through validating model predictions with experimental results, it was found that the hydrolytic degradation of thiol-ene hydrogels was not only governed by ester bond hydrolysis, but also affected by the degree of network crosslinking. In an attempt to manipulate network crosslinking and degradation rate of thiol-ene hydrogels, different macromer contents and peptide crosslinkers with different amino acid sequences were used. A chymotrypsin-sensitive peptide was also used as part of the hydrogel crosslinkers to render thiol-ene hydrogels enzymatically degradable. The second objective of this thesis was to develop a visible light-mediated thiol-ene hydrogelation scheme using a type II photoinitiator, eosin-Y, as the only photoinitiator. This approach eliminates the incorporation of potentially cytotoxic co-initiator and co-monomer that are typically used with a type II initiator. In addition to investigating the gelation kinetics and properties of thiol-ene hydrogels formed by this new gelation scheme, it was found that the visible light-mediated thiol-ene hydrogels were highly cytocompatible for human mesenchymal stem cells (hMSCs) and pancreatic MIN6 beta-cells. It was also found that eosin-Y could be repeatedly excited for preparing step-growth hydrogels with multilayer structures. This new gelation chemistry may have great utilities in controlled release of multiple sensitive growth factors and encapsulation of multiple cell types for tissue regeneration.