Дисертації з теми "Protein functionalization"

Ovarian cancer is the fifth leading cause of cancer death among women in United States and has an alarming 1.4% (1 in 71) lifetime risk. The lack of overt symptoms and the absence of a reliable screening test to detect ovarian cancer result in over 70% of women being diagnosed after the disease has spread beyond the ovary resulting in a poor prognosis. A key characteristic of ovarian cancer is the ability of tumor cells to evade apoptosis, or programmed cell death contributing to the limitless replicative potential, which is a hallmark of all carcinogenesis. There is conclusive evidence that levels of bcl-2 are elevated in ovarian cancer patients' indication that this protein is an ovarian cancer biomarker. The overall goal of this thesis is to functionalize a substrate for specific, sensitive and cost-effective bcl-2 capture. This surface will ultimately be incorporated into an acoustic wave-based diagnostic device for worldwide point-of-care (POC) ovarian cancer detection. This research looks to assess the capture of this analyte protein on a series of bioconjugated surfaces. For the research to be diagnostically applicable, certain factors reveal themselves as more important than others. Since the surface-bound capture antibody must recognize the bcl-2 protein, it is vital to ensure upright orientation of this specific antibody with high affinity for the analyte. Furthermore once integrated with a nanosensor, the surface will sense a change in the mass on the surface, which requires that the surface is highly resistant to non-specific binding. Bioconjugation techniques were employed to initiate self-assembled monolayers (SAM) of silanes, immobilize antibodies (via amine-crosslinking or direct adsorption of protein A/G) and disperse polyethylene glycol (PEG) reagents to reduce non-specific binding on the glass substrates. 3-aminopropyltrimethoxysilane (3-APTMS) and chlorodimethyloctylsilane (ODMS) were deposited on the surface to create initial hydrophilic and hydrophobic properties on which molecular self-assembly could occur. Testing a variety of assemblies with and without the presence of silanes, amine-crosslinking and PEGylation reagents, the substrate displaying the highest efficacy of bcl-2 capture was revealed. These various surfaces were assessed through contact angle and a novel sandwich enzyme linked immunosorbent assay (ELISA) for sensitivity and specificity of bcl-2 standard capture. The consistently low background and facile assembly of the ODMS based substrate with direct adsorption of protein A/G and the PEGylation reagent, Pluronic, was deemed the best functionalized surface for non-specific recruitment of the bcl-2 protein. The substrate also consistently displayed low signal-to-noise ratio which was of extreme importance in this research to guarantee the prevention of false-positive results when detecting nascent carcinogenic behavior. Elucidation of this substrate assembly is the first step towards the long term objective of this thesis, which is to construct a cost-effective early ovarian cancer detection device which can be implemented at the point-of-care to those who need it the most. This is ultimately expected to dramatically improve health outcomes for females worldwide.

Current strategies for designing biomaterials involve creating materials and interfaces that interact with biomolecules, cells and tissues.  This thesis aims to investigate several bioactive surfaces, such as nanocrystalline diamond (NCD), hydroxyapatite (HA) and single crystalline titanium dioxide, in terms of material synthesis, surface functionalization and characterization. Although cochlear implants (CIs) have been proven to be clinically successful, the efficiency of these implants still needs to be improved. A CI typically only has 12-20 electrodes while the ear has approximately 3400 inner hair cells. A type of micro-textured NCD surface that consists of micrometre-sized nail-head-shaped pillars was fabricated. Auditory neurons showed a strong affinity for the surface of the NCD pillars, and the technique could be used for neural guidance and to increase the number of stimulation points, leading to CIs with improved performance. Typical transparent ceramics are fabricated using pressure-assisted sintering techniques. However, the development of a simple energy-efficient production method remains a challenge. A simple approach to fabricating translucent nano-ceramics was developed by controlling the morphology of the starting ceramic particles. Translucent nano-ceramics, including HA and strontium substituted HA, could be produced via a simple filtration process followed by pressure-less sintering. Furthermore, the application of such materials as a window material was investigated. The results show that MC3T3 cells could be observed through the translucent HA ceramic for up to 7 days. The living fluorescent staining confirmed that the MC3T3 cells were visible throughout the culture period. Single crystalline rutile possesses in vitro bioactivity, and the crystalline direction affects HA formation. The HA growth on (001), (100) and (110) faces was investigated in a simulated body fluid in the presence of fibronectin (FN) via two different processes. The HA layers on each face were analysed using different characterization techniques, revealing that the interfacial energies could be altered by the pre-adsorbed FN, which influenced HA formation. In summary, micro textured NCD, and translucent HA and FN functionalized single crystalline rutile, and their interactions with cells and biomimetic HA were studied. The results showed that controlled surface properties are important for enhancing a material’s biological performance.

Antikörper und Antigen-bindende Proteine, die an Fluorophore, Tracer und Wirkstoffe konjugiert sind, sind einzigartige Moleküle, welche die Entwicklung wertvoller diagnostischer und therapeutischer Werkzeuge ermöglichen. Allerdings ist der Konjugationsschritt sehr anspruchsvoll und trotz intensiver Forschung noch immer ein bedeutender Engpass. Zusätzlich sind Antigen-bindende Proteine oftmals nicht dazu in der Lage, die Zellmembran zu durchdringen und im Zellinneren nicht funktionsfähig. Daher ist ihre Verwendung auf extrazelluläre Targets beschränkt, was eine bedeutende Anzahl wichtiger Antigene vernachlässigt. Beide Limitierungen bilden Kernaspekte dieser Arbeit. Mit Tub-tag labeling wurde ein neuartiges und vielseitiges Verfahren für die ortsspezifische Funktionalisierung von Biomolekülen und Antigen-bindenden Proteinen entwickelt, und so die Palette der Proteinfunktionalisierungen bedeutend erweitert. Tub-tag wurde erfolgreich für die ortsspezifische Funktionalisierung verschiedener Proteine und Antigen-bindender Nanobodies angewendet, die für konfokale Mikroskopie, Proteinanreicherung und hochauflösende Mikroskopie eingesetzt wurden. In einem weiteren Projekt wurden zellpermeable Antigen-bindende Nanobodies hergestellt und somit das schon lange Zeit bestehende Ziel, intrazelluläre Targets durch in vitro funktionalisierte Antigen-bindende Proteine zu visualisieren und manipulieren, erreicht. Hierzu wurden zwei verschiedene Nanobodies an ihrem C-Terminus cyclischen zellpenetrierenden Peptiden unter Verwendung von Expressed Protein Ligation funktionalisiert. Diese Peptide ermöglichten die Endozytose-unabhängige Aufnahme der Nanobodies mit sofortiger Bioverfügbarkeit. Mit Tub-tag labeling und der Synthese von zellpermeablen Nanobodies konnten wichtige Bottlenecks im Bereich der Proteinfunktionalisierung und Antikörperforschung adressiert werden und neue Tools für die biochemische und zellbiologische Forschung entwickelt werden.
Antibodies and antigen binding proteins conjugated to fluorophores, tracers and drugs are powerful molecules that enabled the development of valuable diagnostic and therapeutic tools. However, the conjugation itself is highly challenging and despite intense research efforts remains a severe bottleneck. In addition to that, antibodies and antigen binding proteins are often not functional within cellular environments and unable to penetrate the cellular membrane. Therefore, their use is limited to extracellular targets leaving out a vast number of important antigens. Both limitations are core aspects of the presented thesis. With Tub-tag labeling, a novel and versatile method for the site-specific functionalization of biomolecules and antigen binding proteins was developed expanding the toolbox of protein functionalization. The method is based on the microtubule enzyme tubulin tyrosine ligase. Tub-tag labeling was successfully applied for the site-specific functionalization of different proteins including antigen binding nanobodies which enabled confocal microscopy, protein enrichment and super-resolution microscopy. In addition to that, cell permeable antigen binding nanobodies have been generated constituting a long thought goal of tracking and manipulating intracellular targets by in vitro functionalized antigen binding proteins. To achieve this goal, two different nanobodies were functionalized at their C-terminus with linear and cyclic cell-penetrating peptides using expressed protein ligation. These peptides triggered the endocytosis independent uptake of the nanobodies with immediate bioavailability. Taken together, Tub-tag labeling and the generation of cell-permeable antigen binding nanobodies strongly add to the functionalization of antibodies and their use in biochemistry, cell biology and beyond.

HER2 overexpression in breast cancer tumors predicts lower overall survival. Because of the aggressive nature of HER2 tumors and the association with metastatic disease, the HER2 receptor holds great promise as a therapeutic target in metastatic breast cancer. We are developing small molecule inhibitors that bind to the ATP binding site of the tyrosine kinase domain in order to inhibit tyrosine auto-phosphorylation. This process controls biological pathways that mediate the cell growth. In normal cells this process is highly controlled. We are targeting the modification of the side chain of the hydroxy methyl group of 2-Hydroxy methyl-5,8-dimethoxy-1,4-naphthaquinone. These compounds should inhibit the tyrosine kinase cascade of reactions thereby suppressing the overexpression of HER2 shutting down the tumor growth. The synthesis and characterization of a series of substituted naphthaquinone analogs with different increasing chain lengths will be reported.

Engineering of biofunctional scaffolds to precisely regulate cell behavior and tissue growth is of significance in regenerative medicine. Protein-based biomaterials are attractive candidates for functionalization of scaffold surfaces since the ability to precisely control protein sequence and structure allows for fine-tuning of cell-substrate interactions that regulate cell behavior. In this thesis, a series of de novo proteins for bio-functionalization of interfaces was designed, synthesized, and studied. These proteins are based on a diblock motif consisting of a surface-active, amphiphilic block β-sheet domain linked to a disordered, water-soluble block with a terminal functional domain. Several types of functional domains were investigated, including sequences that act as ligands for cell surface receptors and sequences that act as templates for the growth of inorganic particles. Under moderate temperature and pH conditions, the amphiphilic β-sheet block was shown to have a strong affinity to a variety of scaffold materials and to form stable protein coatings on hydrophobic materials by self-assembly. Moreover, the surface adsorption of the proteins was shown to have minimal impact on the presentation of the functional end domains in the soluble block. For the case of diblocks with the RGDS integrin binding sequence, the capability for mediating cell attachment and spreading was demonstrated via control over ligand density on hydrophobic polymer surfaces. The case of diblock proteins with templating domains for inorganic materials was investigated for two systems. First, hydroxyapatite-binding domains were ligated to the end terminus of the water-soluble block to develop proteins for possible bone regeneration applications. It was demonstrated that the hydroxyapatite-binding domain had strong affinity to hydroxyapatite nanoparticles and was able to induce calcium phosphate mineralization on the surfaces coated with diblock proteins from dilute solutions with Ca2+.and PO43-. Next, a silver-binding domain was ligated to the end terminus to create a diblock protein for potential antimicrobial surface applications. The silver-binding domain was shown to accumulate and reduce silver ions, resulting in the formation of silver nanoparticles on the surfaces functionalized by the protein.

This thesis mainly deals with the development of photochemical approaches to immobilize carbohydrates on surfaces for glycobiological studies. These approaches have been incorporated into a number of state-of-the-art nanobio-platforms, including carbohydrate microarrays, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), atomic force microscopy (AFM), and glyconanomaterials. All the surfaces have displayed good binding capabilities and selectivities after functionalization with carbohydrates, and a range of important data have been obtained concerning surface characteristics and carbohydrate-protein interactions, based on the platforms established. Besides, a variety of non-carbohydrate and carbohydrate-based molecules have been synthesized, during which process the mutarotation of 1-glycosyl thiols and the stereocontrol in 1-S-glycosylation reactions have been thoroughly studied.
QC 20111004

Mestrado em Biotecnologia
Este projeto teve como objetivo desenvolver novas estratégias para funcionalizar nanopartículas de ouro (Au NPs) de forma a detetar a proteína C-reativa (CRP). Assim, foram sintetizadas nanopartículas de ouro esféricas com tamanhos médios de aproximadamente 10 e 40 nm. Posteriormente, a superfície das Au NPs foi modificada utilizando duas abordagens diferentes. Na primeira abordagem as Au NPs estabilizadas com citrato foram funcionalizadas com grupos ácidos carboxílicos pela modificação da superfície com os ligandos: ácido 11-mercaptoundecanóico (MUDA) e ácido mercaptopropiónico (MPA). Posteriormente a citidina difosfocolina (CDP) foi acoplada covalentemente à superfície das Au NPs utilizando 1-etil-3-(3’- dimetilaminopropil)carbodiimida (EDC) para promover a reação. Na segunda abordagem as Au NPs estabilizadas com citrato foram modificadas com um aptâmero específico para a CRP. Estes materiais foram caracterizados utilizando diversas técnicas de análise nomeadamente espectroscopia de UVvis e de infravermelho (FTIR), medidas de potencial Zeta e de diâmetro hidrodinâmico e microscopia eletrónica de transmissão (TEM). Amostras selecionadas de Au NPs modificadas com aptâmero (Au NPs@ssDNA+NaCl+KCl) e de Au NPs bioconjugadas com CDP (Au NPs@MUDA@CDP_40nm e Au NPs@MPA@CDP_10nm) foram utilizadas para o estudo da deteção da proteína: CRP. As Au NPs foram adicionadas a soluções de concentração conhecida (10 - 100 nM). A deteção da CRP foi investigada através da aquisição de espectros de UV-vis. O aumento da razão de agregação, isto é entre a absorvância a 620 nm e a absorvância da banda de ressonância de plasmão de superfície localizada (A620/ALSPR), foi monitorizada ao longo do tempo, indiciando a deteção da CRP para todas as amostras testadas. Para os sistemas Au NPs@MUDA@CDP_40nm e Au NPs@ssDNA+NaCl+KCl foi possível correlacionar a razão A620/ALSPR com a concentração da CRP através de uma relação linear nas gamas de concentração de 20 - 50 nM (R2=0.9425) e de, 20 - 45 nM (R2=0.9382) respetivamente. Os resultados obtidos, embora preliminares, são promissores sendo necessário avaliar em estudos futuros aspetos tais como reprodutibilidade e interferência de outras proteínas em estudos futuros, tendo em vista o desenvolvimento de biossensores válidos para a deteção de CRP
The main goal of this project was to develop novel strategies for the functionalization of gold nanoparticles (Au NPs) aiming the detection of Creactive protein (CRP). Au NPs with an average size of 10 and 40 nm were synthesized. Then, the surface of Au NPs was modified following two different approaches. The first approach consisted on the surface functionalization of citrate stabilized Au NPs with carboxylic acid groups, by surface modification using 11-mercaptoundecanoic acid (MUDA) and mercaptopropionic acid (MPA). The cytidine diphosphocholine (CDP) was covalently attached to the surface of functionalized Au NPs using 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDC) to promote the coupling reaction. In the second approach, citrate stabilized Au NPs were modified with a CRP specific aptamer. These materials were characterized using several analysis techniques namely UV-vis spectroscopy, infra-red spectroscopy (F-T IR), zeta potential and hydrodynamic diameter measurements and transmission electron microscopy (TEM). Selected samples of Au NPs modified with aptamer (Au NPs@ssDNA+NaCl+KCl) and CDP bioconjugated Au NPs (Au NPs@MUDA@CDP_40nm e Au NPS@MPA@CDP_10nm) were used for studying CRP detection. Au NPs samples were added to CRP solutions of known concentration (10-100 nM). The detection was measured through acquisition of the UV-vis spectra. The increase of aggregation ratio between absorbance at 620 nm and the absorbance of localized surface plasmon resonance band (A620/ALSPR) was monitored along time and, indicated that CRP was detected for all the Au NPs samples tested. For the systems Au NPs@MUDA@CDP_40nm and Au NPs@ssDNA+NaCl+KCl it was possible to find a linear correlation between the ratio A620/ALSPR and the CRP concentration, within the concentration range 20 - 50 nM (R2=0.9425) and, 20 - 45 nM (R2=0.9382), respectively. The results obtained are very promising but still preliminary. Further studies are needed to evaluate key aspects such as reproducibility and interference of other proteins, keeping as main objective the development of valid biosensors for CRP detection.

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.

According to a study of the European Commission, approximately one million hips are replaced by prostheses worldwide every year. The interaction of the human body with foreign materials that are subjected to alternating mechanical load in a highly corrosive environment still provides challenges. The main factors affecting prosthesis failure are stress shielding effect and poor osseointegration. In this thesis the problem of prosthesis failure has been approached from the material and from the osseointegration point of view trying to give a global solution to the problem. Niobium and hafnium, which are demonstrated to be totally biocompatible, were used to design a Ti-based alloy. The effect of the alloying elements regarding microstructure and elastic modulus was studied and the best composition was deeply characterized in terms of microstructure, elastic modulus, corrosion resistance and superficial energy. Recombinant fragments of fibronectin were synthesised spanning the cell attachment site and the heparin binding domain which are important for cell viability. These motifs were used to functionalise the surface of the TiNbHf alloy. Two tethering methods were studied: physisorption and silanisation. Silanisation was not used before to immobilise fibronectin recombinant fragments onto metallic substrates and in this thesis, its good performance was demonstrated. In vitro studies were made with each fragment and with different combinations of the fragments, which showed the importance of the heparin binding domain to obtain a cell response equivalent to that of fibronectin in terms of cell adhesion, proliferation and differentiation.
De acuerdo con un estudio de la Comisión Europea, aproximadamente un millón de caderas son remplazadas por prótesis en el mundo anualmente. La interacción del cuerpo humano con materiales externos sujetos a una carga mecánica alternante en un medio altamente corrosivo todavía presenta ciertos desafíos. Los factores que contribuyen principalmente al fallo de una prótesis son el apantallamiento de cargas y la pobre osteointegracion. En la presente tesis el problema de la fallida de prótesis ha sido abordado desde el punto de vista del material y de la osteointegracion en un intento de dar una solución global al problema. El niobio y el hafnio, cuya total biocompatibilidad ha sido demostrada, se han utilizado para diseñar una aleación de titanio. El efecto de dichos aleantes respecto a la microestructura y el módulo elástico ha sido estudiado y la mejor composición ha sido profundamente caracterizada en términos de microestructura, módulo elástico, resistencia a la corrosión y energía superficial. Fragmentos recombinados de fibronectina han sido sintetizados abarcando la zona de adhesión celular y la unión de heparina, las cuales son esenciales para la viabilidad celular. Dichos motivos han sido utilizados para funcionalizar la superficie de la aleación TiNbHf. Dos métodos de unión diferentes han sido estudiados: fisisorción y silanización. La silanización es un método que no se ha utilizado hasta el momento para inmovilizar fragmentos de fibronectina sobre superficies metálicas y en la presente tesis su idoneidad ha sido demostrada. Finalmente, estudios celulares in vitro se han llevado a cabo con cada fragmento y con diferentes combinaciones de ambos, lo cual ha mostrado la importancia de la zona de unión de heparina para obtener una respuesta celular equivalente a la obtenida con la molécula de fibronectina en cuanto a adhesión celular, proliferación y diferenciación.

Etant au quatrième rang des cancers les plus fréquents chez l'homme, le cancer de la vessie représente un enjeu médical important. Pourtant, à ce jour, seuls des traitements chirurgicaux handicapants et/ou chimiothérapiques non spécifiques peuvent être envisagés. Le projet de thèse s'inscrit dans le cadre de la recherche de thérapies ciblées du cancer de la vessie en ayant pour objectif le blocage, au niveau moléculaire et de manière sélective, des voies de signalisation mises en œuvre par la tyrosine kinase Tyro3 au sein des cellules cancéreuses. La mise en évidence de la surexpression de ce récepteur membranaire dans la majorité des tumeurs de vessie et son rôle dans la survie des cellules cancéreuses ont en effet permis de valider Tyro3 comme cible thérapeutique pour ce type de cancers. Le projet peut se diviser en trois parties : le développement de nouvelles méthodologies de synthèse autour du motif imidazo[4,5-b]pyridine, la synthèse d'une librairie de candidats inhibiteurs en utilisant les méthodes mises au point et enfin l'étude des relations structure-activité vis-à-vis de la protéine kinase Tyro3
Bladder cancer is a major medical issue, being the fourth most frequent cancer in men and treatable only with heavy surgery and/or broad-spectrum chemotherapy. This thesis project deals with the discovery of new targeted therapies of bladder cancer by blocking specifically, at a molecular scale in cancer cells, the signaling pathways in which protein kinase Tyro3 is involved. Indeed, its overexpression in most bladder cancers and the major part it plays in cancer cells survival have led to the validation of protein kinase Tyro3 as a therapeutic target for the treatment of bladder cancer. This thesis project can be divided into three main parts: the development of new synthetic methods around the imidazo[4,5-b]pyridine scaffold, the synthesis of a library of compounds using these methods and eventually the study of structure-activity relationships of these compounds versus Tyro3

Thesis (M. S.)--Bioengineering, Georgia Institute of Technology, 2010.
Committee Chair: Chaikof, Elliot; Committee Member: Conticello, Vincent; Committee Member: Jo, Hanjoong. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Le principal objectif de ce travail de thèse était d’étudier l’influence de la fonctionnalisation des protéines par des sucres ou des polyphénols de raisin dans la formulation et le comportement de microsphères de vitamine A. La formulation de différents conjugués issus soit de la réaction de Maillard soit de la complexation des polyphénols sur les protéines a été effectué sur trois matières premières protéiques : les protéines de pois, le caséinate de sodium de lait de vache et la gélatine de type A porcine. Dans une première partie, les caractéristiques et le pouvoir émulsifiant des conjugués ont été étudiés, et ont confirmé le potentiel de stabilisation d’une huile dans le temps. Une seconde partie s’est concentrée sur les observations au microscope électronique à balayage des microsphères et sur une méthodologie d’observation spécifique à ce genre d’échantillon. Une troisième partie a étudié l’influence des fonctionnalisations sur la stabilité de la vitamine A dans le temps, sur sa libération dans des milieux de digestion gastriques et entériques simulés, et sur la libération de géraniol co-encapsulé. La dernière étude a porté sur le potentiel muco-adhésif des microsphères en utilisant une technique d’analyse originale
The main objective of this thesis was to study the influence of functionalization of proteins by sugars or grape polyphenols in the vitamin A microsphere formulation and behavior. The formulation of different conjugated stemming either from the Maillard reaction or from the complexation of polyphenols on proteins was made on three proteins : pea proteins, sodium caseinate and type A gelatin. In a first part, the characteristics and the emulsifying power of the combined were studied, and confirmed the potential of stabilization of oil in the time. A second part was on the observations with scanning electron microscope of microspheres and on the methodology of specific observation in this kind of sample. The third part studied the influence of functionalization on vitamin A stability in the time, liberation on gastric or enteric digestion media, and liberation of co-encapsulated geraniol. The last study concerned the muco-adhesive potential of microspheres by using an original analysis

Afin de rendre les régimes alimentaires occidentaux plus durables, un changement d’alimentation s’impose. Parmi les sources végétales prometteuses, la fèverole (Vicia faba L.) peut être utilisée comme ingrédient à fort potentiel nutritionnel et fonctionnel dans la formulation de produits alimentaires. Les graines de fèveroles sont transformées en ingrédients, qui peuvent à leur tour être modifiés, fonctionnalisés, afin d’être plus adaptés à des applications alimentaires. Ce travail de thèse avait pour objectif de comprendre le rôle des conditions de transformation des ingrédients riches en protéines de fèveroles sur leurs propriétés fonctionnelles et leur flaveur. L’impact des conditions de transformation, choisies pour être réalistes sur le plan industriel, a été étudié en utilisant une approche multi-dimensionnelle et trouver ainsi un compromis favorable à l’expression des propriétés des différents ingrédients. Plus précisément, un concentrât de fèveroles riche en protéines, traité selon un procédé de transformation doux à l’échelle industrielle, a été sélectionné puis modifié par différentes conditions de transformation, i.e. le pH (2, 4, 6,4 et 11), la température (55, 75 et 95 °C) et la durée du traitement (30 et 360 min). Trente-six ingrédients différents ont ainsi été produits. Ceux-ci ont ensuite été utilisés à deux pH différents, 4 et 7, dans des systèmes modèles proches d’applications de type boissons. Au cours de l'utilisation de ces ingrédients, la fonctionnalité des boissons (propriétés moussantes et émulsifiantes), la perception olfactive des produits et la composition en composés volatils et non volatils ont été étudiées. Les résultats montrent que les conditions de transformation sont capables de moduler les propriétés fonctionnelles et olfactives du concentrât de fèveroles, l’analyse étant renforcée par le biais de différents modèles statistiques. Les propriétés des mousses et des émulsions sont principalement gouvernées par le pH d'utilisation des ingrédients. Un pH proche du point isoélectrique des protéines de fèverole (pH 4) n'est pas favorable ni à la stabilité de la mousse, ni à la capacité d’émulsification ou à la stabilité de l'émulsion. Des corrélations entre les propriétés fonctionnelles et les propriétés physico-chimiques ont été mises en évidence et s’expliquent par les propriétés des protéines. Par ailleurs, la flaveur est fortement influencée par les conditions de modification et d'utilisation des ingrédients, en particulier par le pH. Selon les modifications du concentrât initial, en conditions douces ou sévères, la perception peut être modifiée, pour évoluer d’odeurs vertes à des odeurs cuites, tandis que les conditions d'utilisation des ingrédients (pH) peuvent conduire à des perceptions « douces » ou rances. La composition en composés volatiles de l’espace de tête des ingrédients mis en suspension montre la présence de nombreux aldéhydes et alcools. L'oxydation des lipides apparaît importante dans la génération de composés volatiles, de même que des réactions de dégradation des protéines, des sucres et des caroténoïdes. Les propriétés physico-chimiques et sensorielles des composés à l’origine du potentiel antioxydant, du goût (amertume et astringence), de la couleur et des effets antinutritionnels ont également été étudiés. Les composés phénoliques et les saponines se sont avérés significativement impactés par les conditions de transformation mises en œuvre lors de la modification des ingrédients, en particulier par le pH. Les profils en composés phénoliques et en saponines obtenus à l’issue des traitements acide et alcalin (pH 2, 4 et 11) apparaissent ainsi très distincts de celui obtenu par un traitement effectué sans ajustement de pH (pH 6,4). Des études complémentaires menées à pH 6,4 indiquent que cette différence serait liée à une question d’extractibilité variable des composés étudiés et/ou à une réactivité plus ou moins élevée selon les conditions de transformation
The growing population is demanding new healthy, sustainable solutions for foods and beverages. Fava bean (Vicia faba L.) is a promising plant source that can provide nutritional and functional ingredients for different food applications. Fava bean is processed to form ingredients and they can be further modified to render them fit for food applications. This PhD work aimed to understand the role of processing conditions on functional and flavor properties, and apply this understanding to produce and use fava bean protein-rich ingredients. It investigated the effects of certain industrially relevant process conditions using a cross-dimensional approach to find the right kind of compromise between different ingredients properties. To be precise, a very gently processed fava bean protein rich concentrate was industrially procured, which was then modified by process conditions such as pH (2, 4, 6.4 and 11), temperature (55, 75 and 95 °C) and treatment duration (30 and 360 min) to produce 36 different ingredients. These were further utilized at two pH (4 and 7) in systems close to beverage applications. During ingredient utilization, beverage functionalities (foam and emulsion) along with odor perception and non-volatile compounds were investigated for all ingredients as a function of process conditions.Results showed that process conditions were able to drive functional and flavor properties of the fava bean concentrate, strengthened by different statistical models. Foam and emulsion properties were predominantly governed by the pH during ingredient utilization. In general, utilization pH around the isoelectric point of fava proteins (pH 4) was not suitable for foam stability, emulsion capacity nor emulsion stability. Strong correlations between functional and physico-chemical properties were identified and explained by protein properties. In addition, flavor was heavily driven by the modification and utilization conditions, especially the pH.From gentler to vigorous process conditions, perception can be modified from more green to more cooked flavors, whereas different conditions of application (e.g. pH) can modulate between “sweet” and rancid perceptions. Considering volatiles composition, aldehyde signals were primarily detected in ingredient suspensions head-space. But furanoids, terpenoids, alcohols and ketones signals had the next higher contribution for modifications at pH2, 4, 6.4 and 11 respectively. Lipid oxidation was deemed important in generating volatiles, along with other reactions including proteins, sugars and carotenoids degradation. Going deeper into understanding of physico-chemical and sensory properties, determinants of antioxidant potential, taste (bitterness and astringency), color and even anti-nutritional effects were also investigated. Phenolic compounds (flavan-3-ols, flavones, flavonols, hydroxycinnamic acids) and saponins were significantly impacted by process conditions during ingredient modification, especially by pH. For phenolic compounds, acidic and alkaline conditions (pH 2, 4 and 11) were highly distinct compared to the non-pH adjusted process (pH 6.4) in changing the phenolic and saponin profiles of the ingredients. When looked closely at non-pH adjusted processes, their variability due to increasing degree of processing seemed to be either a function of their variable extractability and/ or their reactions involving their structural rearrangement.Thus, process conditions played an important role in fava bean ingredient properties, and this work opens up new arena for inter-disciplinary study based on nutritional (anti-oxidant and anti-nutritional aspects), sustainability (life cycle assessment), functionality (gelation) and sensory (texture, sweetness, bitterness) considerations of fava bean as potential ingredients for industrial food applications

Thesis (Ph.D.)--University of California, San Francisco with the University of California, Berkeley, 2010.
Source: Dissertation Abstracts International, Volume: 71-02, Section: B, page: . Adviser: Francis C. Szoka.

Before any novel biochemical device is fabricated, one needs to know that the technology and methodology being used are the most efficient. In the case of immobilised proteins, one needs to know that the method used will not harm the protein and that the conjugation technique used facilitates the highest binding efficiencies for the protein in question. Three methods from literature (non-specific binding, non-covalent binding and covalent binding) were used to conjugate bovine serum albumin (BSA) to single-walled carbon nanotubes (SWNTs). Efforts to use simple methods for protein concentration determination were hampered by the strong absorbance and interference from the nanotubes themselves. FITC tagged BSA was then used to investigate the conjugation methods. This technique was also subject to interference from the nanotubes when they were present. However, the subtraction of the amount of BSA removed from the nanotubes during washing was found to be accurate enough to compare the different conjugation techniques used. This showed that non-specific binding was the best method for conjugation, with approximately 79% of the original BSA remaining on the nanotubes after eight washes. The non-covalent and covalent method showed efficiencies of 54 and 60% respectively. A novel method for protein binding was investigated. This involved using a short DNA oligonucleotide as a tether for binding. The protein concentration was investigated as before using fluorescent BSA. This showed that approximately 88% of the BSA originally added remained on the nanotubes after washing. This value is higher than the other methods investigated and therefore this was the best technique. In addition to BSA conjugation, the enzyme urease was attached using non-specific binding and non-covalent conjugation. An attempt was made to determine the amount of urease bound through an assessment of its catalytic activity. This investigation was unable to accurately determine the amount of urease present, but did show there to be a difference in the amount of urease bound to SWNTs using either non-specific binding and non-covalent binding. The investigation of the best method for protein immobilisation in this thesis has shown that the novel DNA mediated conjugation is the most efficient, closely followed by the simplest method, non-specific binding. Both of these methods can allow the protein to remain structurally intact, an important factor to consider in the creation of novel devices.

Le remplacement des additifs controversés dans les matrices céréalières constitue en enjeu majeur pour répondre aux attentes des consommateurs. Les poudres à lever sont des ingrédients fonctionnels permettant l’obtention de produits biscuitiers alvéolés selon les modes de fabrication industriels. Leur incorporation dans la pâte à biscuits conditionne l’expansion des pâtons lors de l’étape de cuisson. Dans ces travaux, nous avons considéré deux pâtes céréalières avec des taux d’hydratation différents qui déterminent les voies d’incorporation de gaz dans chacune d’elles, avec l’objectif de totalement supprimer les poudres à lever. Dans une pâte à biscuits laminée faiblement hydratée, l’étude a considéré l’utilisation de levures boulangères pour substituer les poudres à lever. La configuration du réseau de gluten module les propriétés élastiques de la pâte et sa capacité à s’étirer pour permettre l’expansion des biscuits à la cuisson. Dans une pâte jaune foisonnée, l’incorporation d’air repose sur la formation d’une mousse stable en même temps que le dégagement gazeux induit par les poudres à lever. L’élimination des poudres à lever, dans cette matrice, a été possible grâce à l’utilisation de protéines végétales fonctionnalisées via différents traitements (physiques ou enzymatiques). Les propriétés interfaciales des protéines de la pâte déterminent leur capacité à stabiliser les bulles d’air qu’elle contient. Celles-ci ont été étudiées par tensiométrie et rhéologie interfaciale. Une approche par plan d’expériences a été implémentée pour optimiser les fonctionnalités et ainsi garantir une alvéolation régulière dans les biscuits
The replacement of controversial additives in cereal matrices represents a major challenge to meet consumers’ expectations. Leavening agents are functional ingredients that are required to obtain porous biscuit products according to industrial manufacturing methods. Their incorporation into biscuit dough determines the expansion of dough pieces during the baking stage. In this work, we considered two cereal doughs with different hydration levels that determine the gas incorporation pathways, aiming to completely suppress the need for leavening agents. In a low-hydration laminated biscuit dough, the study considered the use of baker's yeast as a substitute for leavening agents. The configuration of the gluten network conditions the dough elasticity and its ability to stretch to allow the biscuits to expand during baking. In a sponge drop (whipped) dough, air incorporation relies on the formation of a stable foam simultaneously with the gas release induced by the leavening agents. The removal of leavening agents from this matrix was enabled by using functionalized plant proteins through various treatments (physical or enzymatic). A design of experiments approach was implemented to optimize functionalities and thus, ensure the obtention of biscuits with a uniform crumb structure. During this process, the interfacial properties of the dough proteins determine their ability to stabilize the air bubbles in the matrix. These were studied using tensiometry and interfacial rheology

L'objectif de cette thèse est de créer de nouvelles surfaces nanostructurées avec des revêtements bioactifs et d'étudier leurs propriétés physico-chimiques afin de développer de meilleurs modèles d'implants dentaires et d'optimiser leur ostéo-intégration. Cette fonctionnalisation a été réalisée en deux étapes ; on a commencé par la nano structuration de la surface de TiO2 par anodisation pour créer des sites réactifs sur les bords extérieurs des nanotubes qui agissent comme des points d'ancrage du revêtement bioactif et améliorent le verrouillage mécanique entre le revêtement et le substrat. Ensuite, la modification chimique est réalisée par revêtement de la surface nanostructurée avec des revêtements bioactifs de phosphate de calcium (CaP) et phosphate de calcium dopé par strontium (Sr.CaP). Ce revêtement a été réalisé par électrodéposition pulsée. La caractérisation physico-chimique par MEB, XPS et IR a montré que le dopage avec Sr favorise un composé non-apatitique similaire à DCPD ou DCPA (Dicalcium Phosphate Dihydrate ou Anhydrous), tandis que le revêtement de CaP non-dopé ressemble à un composé d'apatite amorphe ACP. L'addition de strontium s'offre le double avantage de favoriser les mécanismes de la croissance cellulaire et d'obtenir une phase inorganique avec de bio-performances meilleurs que les composés apatitiques. Nous avons également évalué les propriétés d'adsorption de ces surfaces fonctionnalisées en étudiant l'adsorption des protéines (BSA).Cette adsorption a été réalisée sur nanotubes fonctionnalisés vierges, nanotubes enrobés avec CAP et CAP dopé Sr et elle a été évalués selon le temps de déposition et la valeur du pH de la solution qui affecte la charge de la protéine et de la surface. L'évaluation cinétique et structurelle révèle diffèrent géométries d'adsorption en fonction du pH, du temps d'adsorption et aussi en fonction de la nature chimique de la surface. Ces résultats de l'adsorption et conformation de protéine forment une base de données pour comprendre et contrôler ses activités et réactions avec le vivant lorsqu'elle est utilisée dans le system des implants dentaires
The objective of this thesis is to create new nanostructured surfaces with bioactive coatings and to study theirs physicochemical properties in order to develop better dental implants designs and promote their osseointegration. This functionalization was performed in two steps; starting by the nanostructuration of TiO2 surface by anodisation to create reactive sites on the edges of titanium nanotubes which acts as points of “attachment" to bioactive coatings. The second step was the surface chemical modification by coating the nanostructured surface with bioactive coatings of calcium phosphate (CaP) and strontium doped calcium phosphate (Sr.CaP). This coating was performed by pulsed electrodeposition. The physicochemical characterization by XPS, SEM and IR showed that doping with Sr promotes a non-apatitic compound similar to DCPD or DCPA (Dicalcium Phosphate Dihydrate or Anhydrous), while undoped CaP coating looks like an amorphous apatite-like compound ACP. The addition of strontium has the double advantage of optimizing the cellular multiplication and of giving an inorganic phase with bio-performance better than apatitic compounds. We also evaluated the adsorption proprieties of these functionalized surfaces by investigating the adsorption of proteins (BSA). This adsorption was performed onto tblank nanotubes, nanotubes coated with CaP and Sr doped CaP and evaluated according to deposition time and to the pH value of the solution that affect both protein and surface charge. The kinetic and structural evaluation reveals different adsorption geometries according to pH and adsorption time and also according to the chemical nature of surface. Such results of protein adsorption and conformation may form a database to understand and control protein activities and reactions with living body when used for dental implants system

This thesis is focused on synthesis of quantum dots (QDs) for protein detection. It comprises three parts. The first part summaries the theory of QDs, their synthesis, functionalization, interactions and applications in medicine. In the second part synthesis of CdTe/ZnS core/shell QDs modified by glutathione (GSH) is described, followed by the conjugation with biomolecules BSA and IgG. Several coupling agents such as EDC with NHS and CDI were used. In the last part, the final products were characterized by fluorescence spectroscopy and capillary electrophoresis. The results show the dependence of the fluorescence intensity of the QDs on pH range, concentration of BSA and IgG concentrations using different crosslinkers.

Dans le domaine pharmaceutique, la voie orale demeure la voie d’administration de prédilection, car plus simple et mieux acceptée par les patients. Cependant, ce mode d’administration pose problème pour de nombreux principes actifs (PA) présentant une faible solubilité, une faible perméabilité et/ou une instabilité dans l’environnement gastro-intestinal. Leur micro-encapsulation dans des matrices polymériques peut permettre d’y répondre, notamment si les microparticules générées résistent aux environnements rencontrés lors du tractus gastro-intestinal et jouent alors un rôle protecteur, tant pour le principe actif que pour les muqueuses rencontrées. La recherche de nouveaux excipients, issus des agro-ressources tels que les polymères naturels, est en plein essor. Les protéines végétales, grâce à leurs propriétés fonctionnelles telles qu’une bonne solubilité, une viscosité relativement basse, et des propriétés émulsifiantes et filmogènes, représentent des candidats privilégiés. De plus, la grande diversité de leurs groupements fonctionnels permet d’envisager des modifications chimiques ou enzymatiques variées. L’objectif de ce travail était d’étudier l’intérêt de la protéine de soja en tant que matériau enrobant de principes actifs pharmaceutiques destinés à la voie orale, et plus particulièrement en tant que candidat pour l’élaboration de formes gastro-résistantes. Un isolat protéique de soja (SPI) été utilisé comme matière enrobante et l’atomisation comme procédé. L’ibuprofène, anti-inflammatoire non stéroïdien, a été choisi comme molécule modèle du fait de sa faible solubilité nécessitant une amélioration de sa biodisponibilité, et de ses effets indésirables gastriques nécessitant une mise en forme entérique. Deux modifications chimiques des protéines (l’acylation et la succinylation) ont été étudiées dans le but de modifier la solubilité de la protéine de soja. Ces modifications ont été effectuées dans le respect des principes de la Chimie Verte, notamment en absence de solvant organique. Les microcapsules obtenues par atomisation ont été caractérisées en termes de taux et efficacité d'encapsulation, morphologie et distribution de tailles des particules, état physique du PA encapsulé et capacité de libération en milieu gastrique et intestinal simulé. Les résultats obtenus ont permis de valider l’intérêt des modifications chimiques de la protéine de soja pour moduler les cinétiques de libération d’actif. Les modifications chimiques sont apparues particulièrement adaptées pour l’encapsulation de principes actifs hydrophobes, et ont permis de l’obtention de cinétiques de libération d’ibuprofène ralenties à pH acide (gastrique). La dernière partie de ce travail a permis de valider cette dernière hypothèse par la réalisation de formes gastro-résistantes sur le modèle des comprimés MUPS (multiple unit pellet system). Les résultats de ce travail exploratoire démontrent que les protéines de soja, associées à un procédé de mise en forme multi-particulaire couplé à de la compression directe, peuvent constituer une alternative biosourcée, respectueuse de l’environnement (manipulation en solvant aqueux, temps de séchage et étapes de compression réduits) et sûre à l’enrobage utilisé dans les formes gastro-résistantes traditionnelles
In the pharmaceutical field, the oral route remains the preferred route of administration because it is simpler and better accepted by patients. However, this mode of administration is problematic for many active pharmaceutical ingredients (API) with low solubility, low permeability and/or instability in the gastrointestinal environment. Their microencapsulation in polymeric matrices can make them able to respond to these factors, especially if the microparticles generated resist the environments encountered during the gastrointestinal tract and then play a protective role, both for the API and for the mucous membranes encountered. The search for new excipients, from agroresources such as natural polymers, is booming. Vegetable proteins, thanks to their functional properties such as good solubility, relatively low viscosity, and emulsifying and film-forming properties, are preferred candidates. In addition, the great diversity of their functional groups makes it possible to envisage various chemical or enzymatic modifications. The aim of this work was to study the interest of soy protein as a coating material for API intended for the oral route, and more particularly as a candidate for the development of gastro-resistant forms. A soy protein isolate (SPI) was used as a coating material and the atomization as a process. Ibuprofen, a nonsteroidal anti-inflammatory drug, was chosen as a model molecule because of its low solubility requiring an improvement in its bioavailability, and its gastric side effects requiring an enteric shaping. Two chemical modifications of proteins (acylation and succinylation) have been studied in order to modify the solubility of the soy protein. These modifications were carried out in accordance with the principles of Green Chemistry, especially in the absence of organic solvent. The microcapsules obtained by spray-drying were characterized in terms of rate and encapsulation efficiency, morphology and size distribution of the particles, physical state of the encapsulated API and capacity of release in simulated gastric and intestinal medium. The results obtained validated the interest of the chemical modifications of the soy protein to modulate the release kinetics of API. The chemical modifications appeared particularly suitable for the encapsulation of hydrophobic active ingredients, and allowed to obtain ibuprofen release kinetics decreased to acidic pH (gastric). The last part of this work allowed to validate this last hypothesis by the realization of gastro-resistant forms on the model of MUPS tablets (multiple unit pellet system). The results of this exploratory work demonstrate that soy protein, combined with a multiparticle shaping process coupled with direct compression, can be a biosourced, environmentally friendly alternative (aqueous solvent handling, drying and compression steps reduced) and confident to the coating used in traditional gastroresistant forms

Les protéines extraites des végétaux sont des matériaux relativement peu coûteux, non toxiques, biocompatibles et biodégradables. Elles représentent une bonne alternative aux protéines d’origine animale et aux polymères dérivés du pétrole. Dans le cadre de cette étude, les protéines extraites de graines de soja et de tournesol ont été utilisées en tant que matériaux enrobants pour la microencapsulation de la matière active hydrophobe (α-tocophérol) ou hydrophile (acide ascorbique) par le procédé d’atomisation. Les protéines de soja sont largement utilisées dans les applications alimentaires et non-alimentaires, notamment en microencapsulation. Elles sont donc étudiées dans ce travail comme matériau enrobant de référence. Les protéines de tournesol n’ont quant à elles pas d’application industrielle concrète, si ce n’est sous la forme de tourteaux dans l’alimentation animale. C’est pourquoi il nous semble pertinent de trouver des nouvelles voies de valorisation pour ce coproduit d’origine agricole. Plusieurs modifications des protéines, telles que l’hydrolyse enzymatique, l’acylation, la réticulation enzymatique et la cationisation ont été étudiées dans le but d’améliorer les propriétés encapsulantes du matériau enrobant. Dans le contexte de la chimie verte, toutes les modifications ont été effectuées sans utilisation de solvants organiques ni de catalyseurs chimiques. L’influence des modifications chimiques et enzymatiques des protéines, et des paramètres du procédé (pression d’homogénéisation, ratio matériau enrobant/matière active et concentration en protéines) sur les différentes caractéristiques des préparations liquides et des microparticules (viscosité, taille des gouttelettes dans le cas des émulsions, morphologie et taille des microparticules), ainsi que sur les paramètres liés au procédé d’atomisation (rendement et efficacité de microencapsulation) a été particulièrement étudiée au cours de ce travail. Les résultats obtenus confirment que l’extrait protéique de tournesol est tout à fait pertinent comme matériau enrobant et permet d’obtenir des efficacités de microencapsulation significativement plus élevées par rapport à celles obtenues avec l’extrait protéique de soja
Proteins extracted from vegetables are relatively low-cost, non-toxic, biocompatible and biodegradable raw materials. They represent a good alternative to animal-based proteins and petroleum-extracted polymers. In this study, proteins derived from soybean and sunflower seeds were used as wall materials for microencapsulation of hydrophobic (-tocopherol) or hydrophilic (ascorbic acid) active material by spray-drying technique. Soybean proteins are widely used in food and non-food applications, especially in microencapsulation. They were studied in this work as wall material of reference. Sunflower proteins are not actually used in industrial application, but only in the form of oil-cake for animal feeding. That’s why new ways of valorization of this agricultural by-product should be investigated. Several proteins’ modifications such as enzymatic hydrolysis, acylation, cross-linking and cationization were studied in order to improve encapsulating properties of wall material. In the context of green chemistry, all the modifications and preparations were performed without use of organic solvents and chemical catalysts. The effect of protein chemical and enzymatic modifications, and process parameters (homogenization pressure, wall/core ratio and protein concentration) on different characteristics of liquid preparations and microparticles (viscosity, emulsion droplet size, microparticle size and morphology) and on parameters related to the spray-drying process (yield and efficiency of microencapsulation) was particularly investigated in this study. The obtained results confirmed that sunflower proteins are quite suitable as encapsulating agent and provide the microencapsulation efficiencies significantly higher compared to those obtained with soy proteins

Ce travail de recherche est consacré à l’ingénierie d’un nouveau nanobiohybride à base de nanorubans de titanates pour la médecine régénérative. Dans un premier temps, les nanorubans ont été synthétisés par traitement hydrothermal et leurs caractéristiques morphologiques, structurales et chimiques ont été définies. Une caractérisation fine par différentes techniques de microscopie électronique à transmission a notamment permis de déterminer leur épaisseur; dimension qui n’avait encore jamais été mesurée. Par la suite, les nanorubans de titanates ont été fonctionnalisés par différents PEG hétérobifonctionnels préalablement synthétisés au laboratoire. Ces polymères présentent à l’une de leurs extrémités des groupements fonctionnels spécifiques pouvant se coupler à de nombreuses molécules biologiques. Des peptides de type collagène contenant des sites de reconnaissance cellulaire ont alors été greffés sur ces extrémités. Le nanobiohybride ainsi formé devra permettre l'adhésion et la prolifération des cellules favorisant in fine la cicatrisation et la régénération tissulaire. Pour évaluer les propriétés biologiques du nouveau nanobiohybride, la cytoxicité et le pouvoir agrégeant des nanorubans de titanes ont été déterminés par des tests MTT, réalisés sur deux populations de cellules (cardiomyocytes et fibroblastes) et par des tests d’agrégation plaquettaire (sang humain). Enfin, dans le cas d’une utilisation pour favoriser le processus de cicatrisation, le nouveau nanobiohybride a été formulé sous forme d’un hydrogel d’alginate de sodium permettant une application directe sur les tissus lésés. Pour confirmer l’intérêt de cette formulation galénique, des premiers tests in vivo ont été réalisés
This research work is devoted to new nanohybrid engineering composed of titanate nanoribbons for regenerative medicine. Over a first phase, nanoribbons were synthesized by hydrothermal treatment and their morphological, structural and chemical features were defined. A fine characterization by means of different techniques of transmission electron microscopy mainly enabled to determine their thickness; dimension which had never been measured so far. Subsequently, titanate nanoribbons were functionalized by different home-made heterobifunctional PEG. Those polymers present at one of their extremities specific functional groups being able to couple with numerous biological molecules. Some collagen type peptides containing cellular recognition sites were grafted onto those extremities. The so-formed nanobiohybrid will permit cellular adhesion and proliferation favouring in fine tissue healing and regeneration. To evaluate new nanohybrid biological properties, titanate nanoribbons cytoxicity and aggregating power were determined by MTT tests, performed on two cell populations (fibroblasts and cardiomyocytes) and platelet aggregation tests (human blood). Finally, when used to promote healing process, the new nanobiohybrid was formulated in the form of sodium alginate hydrogel permitting a direct application on damaged tissues. To confirm the interest of this galenic form, initial in vivo tests were realized

Recherche d'adsorbants synthétiques constitués de polymères fonctionnels capables d'adsorber les anticorps anti viii: c dans des systèmes d'épuration plasmatique. Substitution de fonction sulfonate et sulfamion d'un acide aminés ou de deux acides amines sur un polystyrène

Les biosalissures marines représentent l’accumulation indésirable d’organismes biologiques sur les surfaces de structures immergées dans l’eau de mer. Malheureusement, ce phénomène naturel a de sérieuses conséquences sur les plans économiques, environnementaux et matériels. Depuis l’interdiction de certains biocides dans les peintures antisalissures (en particulier le TBT en janvier 2008) à cause de leur toxicité envers des espèces marines non-ciblées et de leur accumulation dans l’environnement marin, la recherche a mis l’accent sur le développement de nouveaux revêtements antifouling efficaces,durables et respectueux de l’environnement sans relargage d’espèces toxiques. Ainsi, les travaux de cette thèse portent sur la fonctionnalisation de carbone vitreux par des glucides reliés à des systèmes conjugués électrostimulables via un lien triazole pour développer des surfaces à activité antifouling. En effet, ce type de revêtement a été conçu pour intervenir dans les premières étapes du biofouling. Tout d’abord, le glucide, très hydrophile, devrait lutter contre la formation du film conditionnant en s’entourant d’une barrière aqueuse résistante aux protéines. D’autre part, la modification de l’état de charge de la surface en continu par application d’un courant électrique sur le système conjugué électro-actif devrait perturber la colonisation bactérienneretardant l’installation du biofilm marin. Notre étude repose donc sur la synthèse et l’immobilisation d’un ensemble de glucides électrostimulables sur une surface de carbone vitreux par oxydation de l’amine aromatique en milieux organiques et aqueux. Un test microbiologique a été réalisé sur un des revêtements glucidiques en présence de la souche bactérienne TC8 dans les puits d’une microplaque contenant des cellules électrochimiques reliées à un potentiostat. La stimulation électrique de ce revêtement a permis d’améliorer ses propriétés antibactériennes
Marine biofouling represents the undesirable accumulation of biological organisms on the surfaces of structures submerged in seawater. Unfortunately, this natural phenomenon has serious economic, environmental and material consequences. Since the ban of some biocides in antifouling paints (TBT in January 2008) because of their toxicity on the nontargeted marine species and their accumulation in the marine environment, research has focused on the development of new efficient, durable and environmentally friendly antifouling coatings without releasing toxic species. Thus, the work of this thesis deal with the functionalization of glassy carbon surface by carbohydrates linked to an electrostimulable conjugated system via a triazole link in order to develop surfaces with antifouling activity. Indeed, this kind of coating was designed to intervene in the first steps of biofouling. First, the carbohydrate, which is very hydrophilic, should fight against the formation of the conditioning film by surronding itself with an aqueous barrier resistant to proteins. On the other hand, the continuous modification of charge state by applying an electric current to the electroactive conjugated system is expected to disrupt the bacterial colonization delaying the installation of marine biofilm. Our study is therefore based on the synthesis and the immobilization of electrostimulable carbohydrates on a glassy carbon surface by aromatic amine oxidation in organic and aqueous media. A microbiological test was carried out on one of the carbohydrate coatings in the presence of the TC8 bacterial strain in the wells of a microplate containing electrochemical cells connected to a potentiostat. Electrical stimulation of this coating allowed to improve its antibacterial properties

Tese de Doutoramento em Biologia Molecular e Ambiental - Especialidade em Biotecnologia Molecular
Recombinant DNA technology has enabled us to create and produce new genetic (re)combinations otherwise not found in natural proteins or peptides. Recombinant protein-based polymers (rPBPs) arise from this technology as a class of biopolymers based on repetitive blocks of amino acid residues commonly found in structural proteins, such as the VPGVG block from mammalian elastin and the GAGAGS block from silk fibroin. The main objective of this thesis was focused on developing, production and processing of rPBPs, targeting its biomedical application. Antimicrobial Peptides (AMPs) are natural alternatives to common antibiotics which can act as antimicrobial domain when fused with a rPBP sequence. In this work genetic sequences of rPBPs were recombined with different AMPs sequences originating a set of materials in whose physicochemical and biological properties were analysed. Chapter I presents a comprehensive review on the state of the art of AMPs classification, structure and mode of action. A review on the use of AMPs for the creation of antimicrobial polymers is also presented. With respect to the rPBPs, the literature review was mainly focused on elastin-like recombinamers (ELRs) and silk-elastin-like proteins (SELPs). The experimental approaches on the functionalization of ELRs using different AMPs, as well as the material´s characterization are described in chapters II to VI. Chapters VII to IX are focused on the production of the recombinant co-polymer SELP-59-A and the material´s characterization by exploiting distinct processing methods. Chapter II describes the creation, recombinant protein production and nonchromatographic purification of a functional polymer based on the ELR A200 and the ABP-CM4 peptide, originating the new polymer termed CM4-A200. The purified material when processed into free-standing films displayed high antimicrobial activity against bacteria and fungi. The films demonstrated non-cytotoxic effects against human skin cell lines. Furthermore, they remained stable in dry and wet conditions for a wide range of temperatures, without the use of any crosslinking agent. The developed ex vivo protocol using pig skin for antimicrobial testing confirmed the antimicrobial capability of CM4-A200 films for skin applications. In chapter III is presented the work performed with CM4-A200 polymer processed by electrospinning. The fibre mats were characterized and compared to the cast films described in chapter II. Similarly to the films, the fibres retained the antimicrobial activity and revealed no cytotoxic effects. The stability of the material in wet conditions was only maintained at temperatures above 30 ºC, however the fibre mats were more resistant to thermal degradation than the cast films. Structure prediction of the ABP-CM4 peptide and genetic sequence modifications of CM4-A200 construction are presented in chapter IV. In this regard, the antimicrobial activity of the new peptides and polymers suffered no improvements. The processing procedure of the CM4-A200 polymer was modified originating nanotube patterned films, a modification that increases the contact surface area with the microbial cells. As a result, the antimicrobial activity of the CM4-A200 polymer against Staphylococcus aureus was improved. In chapter V the results on the modification of the peptide BMAP-28 sequence and further functionalization of A200 polymer are presented. When processed in cast films the BMAP-18A200 polymer presented high antimicrobial activity against bacteria, yeasts and filamentous fungi. The deletion of the last 10 amino acid residues of BMAP- 28 sequence was essential to achieve the production of the functional polymer based on this peptide. In chapter VI the peptide Hepcidin was utilized to functionalize the A200 polymer creating a highly antibacterial material in the soluble form, a feature not found in the materials described in preceding chapters. Chapter VII describes the production optimization of the recombinant polymer SELP- 59-A in Escherichia coli which resulted in the volumetric productivity of 0.5 g/L. The one-factor-a-time optimization allowed a 2.5-fold improvement over the previous described values. In the two following chapters, distinct techniques were explored to process rPBPs. In chapter VIII, SELP-59-A and SELP-1020-A were processed by electrospinning. The electrospun mats presented size-dependence on the solvent type and polymer concentration and good compatibility for skin cell line proliferation. This study was further extended to solvent-cast films of SELP-59-A, as presented in chapter IX. The films were stable at temperatures until 220 ºC and displayed electric insulating properties. Methanol treatment induced improvement of the mechanical properties and introduction of glycerol as plasticizer improved flexibility of the material. In the final chapter (X) a general discussion of the results described in the precedent chapters is presented as well as the insights on future perspectives. Overall, this thesis provides new insights on the functionalization of rPBPs, the improvement of the biological production and purification procedures, as well as on the application of distinct processing methods. The rPBPs that were created will certainly contribute to fulfil the demand of new antimicrobial materials in particular for advanced biomaterials.
A utilização de tecnologia de DNA recombinante permite a criação e produção de novas (re)combinações genéticas, que de outro modo não se encontram em proteínas ou péptidos naturais. Desta tecnologia surgiram os polímeros recombinantes de origem proteica (rPBPs), uma classe de biopolímeros baseada na repetição de blocos de aminoácidos normalmente encontrados em proteínas estruturais, tais como o bloco VPGVG da elastina de mamífero ou o bloco GAGAGS da fibroína da seda. Esta tese focou-se no desenvolvimento, produção e processamento de rPBPs, tendo como objetivo a sua aplicação biomédica. Os péptidos antimicrobianos (AMPs) são alternativas naturais aos antibióticos de origem química, podendo ser utilizados como domínio funcional aquando da fusão com rPBPs. Neste trabalho, procedeu-se à recombinação de sequências genéticas de rPBPs com diferentes AMPs, originando uma pletora de novos materiais cujas propriedades físico-químicas e biológicas foram analisadas. No capitulo I é apresentada uma revisão do estado de arte sobre a função, modo de ação e estrutura dos AMPs, bem como a sua utilização para a criação de polímeros com atividade antimicrobiana. Relativamente aos rPBPs, a revisão da literatura focou-se maioritariamente nas famílias dos polímeros recombinantes semelhantes à elastina (ELRs) e dos co-polímeros com base na seda e na elastina (SELP). Os capítulos II a VI focam-se na funcionalização de ELRs utilizando diferentes AMPs, bem como na caracterização e processamento dos polímeros obtidos. Os capítulos VII a IX descrevem os trabalhos desenvolvidos com o co-polímero SELP-59-A, mais especificamente na otimização da sua produção e na exploração de diferentes técnicas de processamento. O capitulo II descreve a criação, produção e purificação por meios não cromatográficos de um polímero funcional baseado no ELR A200 e no péptido ABP-CM4, originando o polímero CM4-A200. O material purificado foi processado na forma de filme apresentando alta atividade antimicrobiana contra bactérias e fungos. Os filmes não apresentaram efeitos citotóxicos em linhas celulares humanas de pele. Além disso, mantiveram-se estáveis em condições secas e húmidas, sem o uso de qualquer agente de reticulação. O ensaio ex vivo com pele de porco que foi desenvolvido demonstrou o potencial dos filmes de CM4-A200 para aplicação na pele. O trabalho com o polímero CM4-A200 processado por eletrofiação é apresentado no capítulo III. As fibras criadas foram caracterizadas e as suas propriedades comparadas com os filmes mencionados no capítulo anterior. Tal como nos filmes, as fibras apresentaram atividade antimicrobiana e ausência de efeitos citotóxicos em linhas celulares humanas. As fibras apenas se mantêm estáveis a temperaturas superiores a 30 °C, quando em condições húmidas, contudo, são mais resistentes à degradação térmica do que os filmes. No capítulo IV descreve-se a previsão de estrutura do péptido ABP-CM4 e a introdução de modificações na construção CM4-A200. No entanto, estas modificações foram ineficazes no melhoramento da atividade antimicrobiana. O processamento dos filmes de CM4-A200 foi modificado originando-se um padrão em “floresta de nanotubos”, resultando no aumento da superfície em contacto com as células de microrganismos. Esta técnica resultou numa maior atividade contra a bactéria Staphylococcus aureus. No capítulo V está descrita a modificação da sequência do péptido BMAP-28 e a sua utilização na funcionalização do polímero A200. Quando processado na forma de filme, o polímero BMAP-18A200 apresentou alta atividade antimicrobiana contra bactérias, leveduras e fungos filamentosos. A remoção dos últimos 10 aminoácidos do péptido BMAP-28 resultou na produção do polímero funcionalizado. No capítulo VI, o péptido hepcidina foi utilizado para a funcionalização do polímero A200. Este revelou propriedades antibacterianas na forma solúvel, contrariamente ao observado nos polímeros mencionados nos capítulos anteriores. No Capítulo VII descreve-se a otimização da produção do polímero recombinante SELP-59-A em Escherichia coli, obtendo-se uma produtividade volumétrica de 0,5 g/L. A otimização do método resultou num aumento de 2,5x relativamente ao descrito na literatura. No capítulo VIII, os polímeros SELP-59-A e SELP-1020-A foram processados por eletrofiação. O diâmetro das fibras foi dependente do solvente e da concentração da solução utilizados. O material demonstrou ausência de citotoxicidade em culturas de linhas celulares de pele. No capítulo IX estudou-se o processamento e caracterização de filmes de SELP-59-A. Este material mostrou-se estável até 220 °C, e apresentou propriedades de isolamento elétrico. O tratamento com metanol induziu alterações nas propriedades mecânicas e a composição com glicerol resultou num aumento da flexibilidade do material. No último capítulo (X) procedeu-se à discussão geral dos resultados apresentados e apresentam-se as perspetivas futuras. De um modo geral, esta tese providencia novas soluções para a funcionalização, produção, purificação e processamento de rPBPs. Com este trabalho pretende-se que os rPBPs aqui criados contribuam para responder à procura de novos materiais com propriedades antimicrobianas, e particularmente, na área dos biomateriais avançados.
The work presented in this dissertation was performed in the Centre of Molecular and Environmental Biology (CBMA), Department of Biology, University of Minho and in the BioForge institute, University of Valladolid, Spain. The financial support was given by Fundação para a Ciência e Tecnologia (FCT) by means of a grant, SFRH/BD/75882/2011. Financial support for part of the work was supported by FCT I.P. through the strategic funding UID/BIA/04050/2013

Tese de doutoramento em Engenharia Têxtil
The formation of intramolecular disulphide bonds is critical in the process of protein folding and on the stabilization of protein tertiary structure. Their formation involves the oxidation of two thiol groups that should be correctly paired. The incorrect paring inhibit the protein folding into its native conformation. The rearrangement of incorrectly disulfide bonds on proteins is catalysed in vivo by the protein disulphide isomerase (PDI). This versatile enzyme is able to catalyse the oxidation, reduction and isomerisation of disulfide bonds in a broad range of protein substrates. This dissertation successfully presents the use of PDI for functionalization of cysteine-containing (CC) proteinaceous substrates such as keratin fibres and RNase A microspheres. These approaches take advantage of the presence of thiol moieties and disulphide bonds in these substrates. It was shown that the type of reaction catalysed by PDI can be predicted, by controlling the redox environment. When the active site of PDI is in its oxidized state due to is characteristic potential redox (Eº = -180 mV) an oxidation reaction is catalyzed. When the active site of PDI is transformed to its reduced state (ΔE = - 260 mV), the isomerisation of disulfide bonds is promoted. PDI was able to incorporate CC functional molecules on wool and hair trough disulfide bonds, as suggested by matrix-assisted laser desorption and ionization time-offlight results (MALDI-TOF) analysis. Similarly, PDI increased the affinity of a synthesised keratin-based peptide (KP) towards hair and facilitated the penetration into its cortex. Targeting a biomedical approach, Ribonuclease A (RNase A) was oxidatively attached to the wool surface through disulphide bonds, and PDI was shown to induce its release. Aiming a cosmetic application, KP and other synthesized surfactant-based peptide (SPB) were applied in over-bleached, damaged hair. Both peptides induced an improvement on its mechanical properties and thermal stability. Thus, recovering of the fibre integrity loss during the hair bleaching process was achieved. In the presence of PDI, peptides were linked by disulphide bonds and the thermal stability increased at higher levels. Due to the great properties developed on over-bleached hair after KP application, in the absence of PDI, other type of hair (relaxed hair) was treated with this peptide and the same properties were measured. The relaxing treatment, commonly applied on excessively curly hair, often result on the weakening of the fibre. KP was then applied to this weakened hair and its ability to recover its mechanical and thermal properties was proved. Two different peptide formulations were evaluated. In one formulation KP was dissolved in aqueous solution (WF) while in the other KP was dissolved in organic solvent solution (OF). The last imparted better mechanical and thermal properties to the hair, however, the safety assessment showed that OF is potentially cytotoxic, inhibit cell growth, and genotoxic. The KP itself did not inhibit the cell growth and was found to be non-cytotoxic and non-genotoxic, hence suitable for the application on cosmetic formulations at concentrations up to 0.5 g/L. Showing its ability to act on a broad range of cysteine-containing compounds, PDI was also able to oxidize the thiol groups found in sphere-like particles, recovering their biological function, at the conditions that lead PDI‟s active site on its oxidized state. Using a reducing environment, PDI promoted the released of native RNase A from protein-based microspheres. The research presented in this thesis shows the versatility of PDI to promote diverse functionalization on proteinaceous substrates, resulting in a wide applicability in different areas such as cosmetic, textile and biotechnology. The work was carried out partially in collaboration with a cosmetic company; hence the research included promising biotoolsbased strategies for hair-care product development, especially for the application on several types of damaged hair.
As pontes dissulfídicas são uma característica muito importante para a estabilização da estrutura terciária de proteínas. A formação das mesmas envolve a oxidação de dois grupos tiol, os quais devem estar correctamente ligados. Caso contrário, a proteína perde a sua actividade biológica característica, devido à formação de pontes dissulfídicas nãonativas, e desnatura. In vivo, aquando da formação das proteínas na célula, uma enzima tem uma função muito importante: promover o rearranjo das pontes dissulfídicas nãonativas e prevenir a agregação das proteínas desnaturadas. Esta enzima é a Protein Disulfide Isomerase (PDI), uma isomerase de pontes dissulfídicas que catalisa a oxidação, redução ou isomerisação de uma vasta gama de substratos contendo cisteína na sua constituição. Esta dissertação apresenta com sucesso a aplicação desta enzima na funcionalização substratos proteicos, tais como as fibras queratinosas ou microesferas de Ribonuclease A (RNase A). As pontes dissulfídicas dos substratos, acima mencionados, são as ligações-alvo nesta abordagem. Foi demonstrado que o potencial de redução do centro activo da PDI é uma ferramenta importante na pré-determinação das reacções que esta enzima pode catalisar. Quando o centro activo da PDI está no seu estado oxidado, devido ao seu característico potencial redox (Eº = -180 mV), uma reacção de oxidação é catalisada. Quando o centro activo da PDI é transformado para o seu estado reduzido (ΔE = - 260 mV), a isomerisação das pontes dissulfídicas é promovida. A PDI catalisou a incorporação de moléculas contendo cisteína (CC) em lã e cabelo através de pontes dissulfídicas, sugerido pela análise de espectroscopia de massa (MALDITOF). A PDI facilitou também, a penetração de um péptido de queratina (KP) no córtex da fibra de cabelo e induziu a libertação de uma proteína modelo (Ribonuclease A) da superfície da lã. Com o objectivo de uma aplicação cosmética, o KP e outro péptido, derivado de um surfactante humano (SPB) foram aplicados em cabelo danificado, previamente sujeito a vários ciclos de branqueamento oxidativo. Ambos os péptidos melhoraram as propriedades mecânicas e a estabilidade térmica do cabelo danificado, provando a sua capacidade para recuperar a integridade da fibra. Quando a PDI foi aplicada, os péptidos ligaram-se ao cabelo através de pontes dissulfídicas e a estabilidade térmica aumentou para valores ainda mais elevados. Devido ao efeito renovador que o KP teve sobre o cabelo branqueado, outro tipo de cabelo (cabelo relaxado) foi tratado com este péptido e as mesmas propriedades foram medidas. Os tratamentos de relaxamento em cabelo extremamente encaracolado resultam em enfraquecimento do mesmo. O KP foi por isso aplicado neste tipo de cabelo e a sua capacidade para o melhorar foi provado. Dois tipos de formulações peptídicas foram também testados. Numa formulação o KP foi diluído numa solução aquosa (WF), enquanto na outra o KP foi diluído numa solução contendo solventes orgânicos (OF). Esta última promoveu melhores resultados, contudo, revelou-se potencialmente cytotoxica, genotoxica e inibidora do crescimento celular. Demonstrou-se, todavia, que o péptido em solução aquosa pode ser aplicado em formulações cosméticas até à concentração de 0.5 g/L não manifestando citotóxicidade, genotóxicidade ou inibição do crescimento celular. Demonstrando a sua aptidão para actuar em compostos contendo cisteína, a PDI foi também capaz de oxidar os grupos tiol presentes nas microesferas, recuperando a sua função biológica nas condições que promovem o estado oxidado do seu centro activo. Usando um ambiente mais redutor, promoveu a libertação da proteína nativa das microesferas proteicas. Os resultados apresentados nesta tese demonstram a versatilidade da PDI para promover a funcionalização de substratos proteicos, resultando numa ampla aplicabilidade em áreas distintas como cosmética, têxtil e biotecnologia. O trabalho foi desenvolvido em parceria com uma empresa de cosmética, o que fomentou a procura de estratégias biológicas para o desenvolvimento de novos produtos para cabelo, especialmente para a aplicação em vários tipos de cabelo danificado.
Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/38363/2007

Exploiting the functional diversity of proteins for fundamental research and biotechnological applications requires their functional organization into micro- and nanostructures while preserving their functional integrity to the highest possible level. My PhD research aimed to establish generic techniques based on photolithography which could be used to control the spatial as well as temporal organization of recombinantly expressed proteins on surfaces. My thesis describes in detail four strategies that I developed for achieving this goal. In the first approach a photo-induced Fenton reaction was used to selectively destroy tris(nitrilotriacetic acid) (tris-NTA) moieties on a surface. UV-irradiation through a photomask allowed localized photo-destruction and targeting of His-tagged proteins to non-irradiated regions. Photo-destruction could also be achieved by scanning selected regions with the UV laser of a confocal laser scanning microscope (CLSM) thus allowing flexible creation and modification of protein patterns. The second strategy was based on the photosensitive nitroveratryloxycarbonyl (NVOC) protection group, which was used to cage amine groups on a surface. Sequential uncaging by UV-irradiation through a photomask followed by reactions with biotin and coenzyme A was used to pattern streptavidin and ybbR-tagged proteins into microstructures. In the third approach a photo-fragmentable Histidine peptide was used to block tris-NTA surfaces against binding of His-tagged proteins. UV-irradiation through a photomask or by using a UV laser in a CLSM cleaved the peptide into short fragments which quickly dissociated from the surface due to loss in multivalency. His-tagged proteins could be efficiently targeted into irradiated regions even from a complex cell lysate. Sequential uncaging and immobilization allowed the construction of multiplexed protein patterns with a high degree of temporal control. The fourth strategy used combined peptide tags comprising of a His-tag as well as a Halo- or ybbR-tag to achieve rapid covalent immobilization of recombinant fusion proteins on surfaces functionalized with specific ligands. In combination with a photo-fragmentable histidine peptide as described above, stable spatio-temporal organization of proteins carrying these combined tags was possible. The techniques developed in this thesis enabled the photolithographical micropatterning of recombinant proteins carrying specific peptide or protein tags on surfaces in a functional manner. Owing to the generic nature of immobilization strategies, coupled with the ease of patterning, highly versatile applications of these methods both in fundamental research as well as bio-technological and analytical applications can be envisioned.

Compared to 'conventional' materials made from metal, glass, or ceramics, protein-based materials have unique mechanical properties. Furthermore, the morphology, mechanical properties, and functionality of protein-based materials may be optimized via sequence engineering for use in a variety of applications, including textile materials, biosensors, and tissue engineering scaffolds. The development of recombinant DNA technology has enabled the production and engineering of protein-based materials ex vivo . However, harsh production conditions can compromise the mechanical properties of protein-based materials and diminish their ability to incorporate functional proteins. Developing a new generation of protein-based materials is crucial to (i) improve materials assembly conditions, (ii) create novel mechanical properties, and (iii) expand the capacity to carry functional protein/peptide sequences. This thesis describes development of novel protein-based materials using Ultrabithorax, a member of the Hox family of proteins that regulate developmental pathways in Drosophila melanogaster . The experiments presented (i) establish the conditions required for the assembly of Ubx-based materials, (ii) generate a wide range of Ubx morphologies, (iii) examine the mechanical properties of Ubx fibers, (iv) incorporate protein functions to Ubx-based materials via gene fusion, (v) pattern protein functions within the Ubx materials, and (vi) examine the biocompatibility of Ubx materials in vitro . Ubx-based materials assemble at mild conditions compatible with protein folding and activity, which enables Ubx chimeric materials to retain the function of appended proteins in spatial patterns determined by materials assembly. Ubx-based materials also display mechanical properties comparable to existing protein-based materials and demonstrate good biocompatibility with living cells in vitro . Taken together, this research demonstrates the unique features and future potential of novel Ubx-based materials.

This thesis is about the development of multimodal porous cellular alumina structures (monoliths) by an emulsion-gel casting technique using eco-friendly and inexpensive lipids such as corn oil, castor oil, margarine and their mixtures as the dispersed phase. The monoliths obtained showed good mechanical stability (in terms of compressive strength) despite being porous (up to 60%). The formation of the porous networks was interpreted based on combined droplet coalescence, flocculation and Ostwald ripening effects. The presence of such effects along the emulsion storage time led to changes in their viscoelastic and morphological properties, which were found to correlate with structural descriptors of the monoliths after sintering (e.g. average pore sizes and porosity). Furthermore, the fact that these monoliths had hierarchically distributed pores supposes that there would be paths or channels for fluids to flow through them. Experimental and computational studies were performed to understand the behaviour of fluid flow through the monolith. As per literature, several modelling approaches have been applied to describe experimentally observed flow behaviour in such materials. Morphology plays a key role in determining their hydrodynamic and mass transfer properties. Therefore, a direct computational fluid dynamics (CFD) modelling approach was applied to simulate flow behaviour in these columns. The morphological structure of a fabricated alumina monolith was first reconstructed using 3D X-ray tomography data and, subsequently, OpenFOAM, an open-source CFD tool, was used to simulate the essential parameters for monoliths’ performance characterisation and optimisation, i.e. velocity and pressure fields, fluid streamlines, shear stress and residence time distribution (RTD). Moreover, the tortuosity of the monolith was estimated by a novel method, using the computed streamlines, and its value (~1.1) was found to be in the same range of the results obtained by known experimental, analytical and numerical equations. Besides, it was observed that fluid transport was dominated by flow heterogeneities and advection, while the shear stress at pore mouths was significantly higher than in other regions. The proposed modelling approach, with expected high potential for designing target materials, was successfully validated by an experimentally obtained residence time distribution (RTD). However, alumina itself is a relatively non-reactive material. Therefore, to explore a potential application for the produced monolith a simple, single stage sol-gel synthesis method was used to functionalize the monoliths with (3-Aminopropyl)triethoxysilane (APTES) in an aqueous environment. The nature of the attachment of APTES to the alumina and its distribution through the monolith column was evaluated using characterization methods involving FTIR-ATR, SEM-EDS and XPS measurements. Furthermore, the reaction conditions in terms of catalyst used (acid or base) and temperature were adjusted and, separately, a factorial experimental design was applied to elicit the interdependent influence of humidity, number of APTES coating layers and precursor concentration on the silanization of alumina. The reaction was found to be optimum at basic pH and a temperature of 80˚C. Optimally functionalized monoliths with highest amine density of 166 μmol/g of the column were obtained with a single coat using 2M APTES solution, and at 100% humidity. Finally, experiments were carried out to understand the protein interactions with the produced amine functionalized alumina monolithic columns. Bovine Serum Albumin (BSA) was used as the model protein. Studies were carried out at varied BSA concentrations (0.5 to 10 mg.mL-1) to understand the interaction behaviour between the protein and the column. It was found that at lower concentrations there appeared to be stronger binding. At higher BSA concentrations, due to the formation of aggregates, the interaction appears to be a multi-layered physical adsorption. Dynamic light scattering measurements further confirmed the presence of protein aggregation phenomena at higher protein concentrations due to the contact of the protein solution with the column. From these inferences, appropriate strategies were used to bind Protein G to the column - a maximum of 1.43 mg Protein G/g of monolith (29% by mass of column) was bound. Finally, a binding-elution experiment using bovine immunoglobulin G was conducted and it was found that 73.4% (IgG/Protein G) could bind to the column and 86% of the bound IgG could be eluted using an appropriate buffer. This proved the potential of the amine functionalized monolith for further application as an Affinity Chromatography medium.

博士
國立臺灣大學
生化科學研究所
107
Fluorescent nanodiamonds (FNDs) containing nitrogen-vacancy (NV) defects as light emitters have been extensively used as contrast agents for bioimaging due to their superior optical properties, such as high photostability. Forming aggregates in biofluids and lacking specific targeting ability, however, have significantly impeded their applications in specific protein labelling and imaging. In this thesis, two strategies were developed to modify and functionalize FNDs to overcome existing limitations. The first approach, lipid encapsulation method has the advantages of simple manipulation and time-effective. Desired functional groups can be added onto FND surface through minor changes in the lipid composition. Alkyne-modified hyperbranched polyglycerol (alkyne-HPG) grafting by ring opening reaction is the other approach for functionalization of FNDs. Although this approach has longer processing time, the coating layer is much more stable because of the formation of covalent bonds between the coating layer and particles. Both coatings endow FNDs with not only high dispersity in physiological medium but also specific targeting ability of cell membrane proteins. By combining the exclusive optical features (e.g., chemical inert), biotinylated lipid coated FNDs (bL-FNDs) successfully simplified the complicated protocol to localize CD44 antigens on cell surface by correlative light electron microscopy (CLEM). A thorough literature search reveal that FND is to date, the only carbon nanomaterial having the ability to act as a dual contrast agent for CLEM. Moreover, taking advantage of magneto-optical property of NV- centers, highly sensitive and accurate quantification of CD44 antigens on cell surface with 35 nm of bL-FNDs was accomplished. Finally, high temporal and spatial resolution of continuous long-term observation of integrins α5 was achieved with alkyne-HPGFNDs. The superb photostability (no photo-bleaching and -blinking) of FNDs allows for the detailed transportation route of integrins α5 to be studied through short- and long-term observation, which cannot be viewed by any other dye molecules or quantum dots. To sum up, two reliable surface functionalization methods for FNDs was successfully demonstrated in this thesis. These novel FNDs shorten the gap between light and electron microscopy and serve as a platform for continuous long-term imaging of membrane protein tracking with high temporal and spatial resolution. In the future, the applicability of other kinds of biohybrid FNDs (e.g., antibody modified HPGFNDs or L-FNDs) may be conducted to further simplify the protocol of FNDs for biolabeling.

Proteins are involved in myriad processes in all organisms. They provide structural support in the membrane and scaffolding of each cell; they aid in the transmission of biochemical signals within and between cells; and they play central roles in combating various disease states. The development of techniques enabling selective and site-specific functionalization of proteins is an active area of investigation, as such modifications are critical to many studies and uses of proteins. For instance, with the addition of a unique reactive handle, a protein may be conjugated to a polymer for the production of protein-based therapeutics exhibiting improved bioavailability. Alternatively, proteins may be attached to slides to prepare diagnostic microarrays, reacted with hydrogels to create functional biomaterials, or decorated with fluorophores for in vivo imaging. Site-specific protein tagging techniques have already contributed greatly to biomedical research and will continue to advance the state of the field.

The focus of my thesis research has been the development of a novel site-specific protein labeling method centered on the eukaryotic enzyme N-myristoyl transferase (NMT). In a process called myristoylation, NMT appends a fatty acid to the N-terminus of numerous substrate proteins. Previous work demonstrated that NMT tolerates a wide range of chemically functionalized analogs of its natural fatty acid substrate. Here, we describe efforts that exploit various features of NMT: its ability to bind and utilize reactive fatty acid analogs, its exquisite selectivity toward its protein substrates, and its orthogonality toward those proteins naturally present in bacteria.

First, in Chapter II, we discuss the development of a model system for NMT-mediated protein labeling in the bacterium Escherichia coli. We synthesized an azide fatty acid analog that can participate in bioorthogonal chemistries, and we prepared two GFP-based substrate proteins, each displaying a recognition sequence derived from a known substrate of NMT. Our experiments indicate that labeling by NMT is site-specific, quantitative, and highly selective for each engineered substrate within the bacterial milieu.

As summarized in Chapter III, the model system was extended to the N-terminal labeling of two neuronal proteins, calcineurin (CaN) and calmodulin (CaM). While CaN is naturally myristoylated, CaM was engineered to achieve labeling by NMT. Experiments with CaN and CaM confirmed that our NMT-based system is quantitative and selective in its labeling of both natural and engineered substrate proteins. Extensive characterization of each protein allowed us to identify constructs that retain wild-type levels of activity even after labeling with the azide fatty acid.

Three of the protein constructs reported in Chapters II and III were utilized for microarray studies, as described in Chapter IV. We achieved rapid surface immobilization of each azide-labeled protein directly from lysate, a significant advantage when considering the time and resources normally required to purify proteins for downstream applications. The experiments and methods summarized in this chapter will be adapted for high-throughput biochemical research with protein microarrays.

Finally, the orthogonality of NMT toward bacterial systems was probed further for the purpose of selective labeling of individual bacterial proteins for live-cell imaging. In addition to identifying an azide fatty acid suitable for such studies, we also selected two bacterial proteins that exhibit interesting functions and localization patterns, and we developed corresponding protein constructs for NMT-mediated labeling. Progress toward the use of NMT for in vivo imaging applications in bacteria is described in Chapter V.

Ultimately, our objective throughout the design and execution of these projects was to create and validate a new technique to achieve site-specific protein labeling. The particular advantages of NMT include its tolerance of reactive fatty acid analogs and engineered substrate proteins, and its lack of interaction with proteins present in the widely used E. coli expression host. We believe that the ideas and results presented in this thesis establish NMT-mediated protein labeling as a valuable tool for addition to the existing set of site-specific protein labeling methods. Development of such methods represents an important and exciting area within the field of modern chemical biology.

Tese de Doutoramento em Engenharia Química e Biológica
Laccase is one of the multi-copper oxidases which can catalyze the oxidation of phenols, aromatic amines and other compounds using oxygen as the terminal electron acceptor. As an environment protecting biocatalyst, laccase shows great potential applications in various fields and industries, including textile industry, pulp and paper industry, environmental pollutant conversion and others In the field of synthesis, laccase catalyzed reactions have been widely investigated. However, the mechanism of laccase catalyzed aromatic compounds still needs to be further explored. The research and development of laccase on the polymer making study meet with requirement of the green chemistry, which will promote the application of the synthetic polymer materials. This PhD thesis intends to explore new strategies and mechanisms for the synthesis of aromatic polymers using different laccase forms and different reactors. Aromatic polymers were prepared using laccase from ascomycete Myceliophthora Thermophila. Different modifications of laccase were designed to improve its performance, including PEGylation and/or immobilization. Molecular modeling simulations were used to predict the geometry and structural changes of laccase. During the polymerization process, three reactors with different energy environment were applied, and the function of high-energy environment was investigated with the assistance of homology modelling and molecular simulation. The polymers obtained were characterized and applied onto different fabrics to achieve multi-functional fabrics. This study will provide fundamental knowledge for the polymer synthesis by laccase, and offer the theoretical and technical support for the establishment of high efficiency laccase-catalyzed synthesis system. Firstly, the polymerization of catechol was conducted using laccase as catalyst. Polyethylene glycol (PEG) was used in both non-covalent and covalent modification of laccase, then the performance of modified laccase was evaluated. The catalytic performance of different laccases was compared in both aqueous and gel phases. The results show that both non-covalent and covalent modification of laccase using PEG (PEGylated laccase) could promote the polymerization and improve the polymerization yield, as well as the degree of polymerization. However, these events were only detected in aqueous state. Molecular simulation shows that the presence of PEG slows down the inactivation of laccase. Later, the immobilization of laccase and PEGylated laccase was performed with epoxy resins as the carrier, using PEG also as linker compound to connect laccase and epoxy resin. The performance of immobilized laccase in aqueous solution for polymerization was explored and the structure of the polymer formed was proposed. After immobilization, the half-life time of laccase was improved, as well as the stability. The MALDI-TOF MS analysis showed that, when epoxy-native laccase, epoxy-PEGylated laccase and epoxy-PEG-laccase were used, the degree of polymerization was enhanced with the presence of immobilized laccase. Secondly, different reactors namely water bath, ultrasonic bath and high-pressure homogenizer were applied to perform the polymerization of catechol. The activity and stability of laccase in those reactors were discussed. The pathway and mechanism of laccase synthesis in high-energy environment were investigated. The polymers were characterized to speculate their structures. The results showed that high-energy environment promote the interaction between enzyme and substrate, incrementing the polymerization yield. The conversion yield when using the ultrasonic bath and high-pressure homogenizer was higher than when water bath was applied. Laccase under ultrasound and high-pressure homogenization showed less stability compared with under normal water bath, however, the polymerization proceeds earlier than this inactivation, thus no obvious negative effect on the synthesis was detected. The performance of native, PEGylated and Epoxy-PEGylated laccases was studied under the high-pressure homogenizer. Their activity and stability were compared and the corresponding polymers produced were evaluated. Both PEGylated laccase and Epoxy-PEGylated laccase showed the greatest catalytic properties and stability. Afterwards, catechol and p-phenylenediamine were polymerized using polyester (PET), cotton and wool as enzyme container in a high-pressure homogenizer using laccase as catalyst. The functionalization of fabrics was achieved via in-situ polymerization of aromatic substrate onto fabric containers. Both polymers, poly(catechol) and poly(p-phenylenediamine), present good thermal stability and resistance to thermal degradation, as well as free radical scavenging ability. Colored polymers were generated which conferred color to the fabrics. The scanning electron microscopy (SEM) observation shows uniform distribution of the polymer on the surface of cotton, wool and PET. All the fabrics reveal color fastness to washing, sunlight and rubbing. The conductivity of fabrics was determined after treatment with poly(p-phenylenediamine) and all the fabrics showed good conductivity. Both polymers are able to confer antimicrobial activity to all the coated fabrics against Gram positive (S. aureus) and Gram-negative (E.coli). The cytotoxicity tests performed on functionalized fabrics revealed that both polymer diffusion and porous fabric structure may affect cell viability, which could be avoided by the adjustment of the polymer concentration. This project aims to study the impact of the enzyme modification and processing conditions on the structure of the multi-functional reaction products, and aims to set up a high efficiency reaction system for laccasecatalyzed synthesis of a wide range of phenolic compounds. The oligomers/polymers obtained are supposed to show different functions to be applied on textile, medical and other areas.
A lacase é uma das oxidases multicobre que é capaz de oxidar compostos fenólicos, aminas aromáticas e outros compostos usando o oxigénio como aceitador de electrões. Sendo um biocatalisador ambientalmente aceite, a lacase mostra elevado potencial em diversas áreas de investigação assim como ao nível industrial, incluindo a Industria Têxtil, a Industria do papel, e na área de conversão de agentes poluentes, entre outros. No campo da síntese, as reações catalisadas pela lacase têm sido intensamente investigadas, no entanto o mecanismo de catálise de compostos aromáticos necessita de ser estudado em maior profundidade. A pesquisa e desenvolvimento da lacase na produção de polímeros atendem aos requisitos da química verde, o que promoverá uma vasta aplicação dos materiais poliméricos sintéticos. Esta tese de doutoramento pretende explorar novas estratégias e mecanismos para a síntese de polímeros usando lacase em diferentes formas e diferentes tipos de reatores. Os polímeros aromáticos foram produzidos usando a lacase do fungo ascomicete Myceliophthora Thermophila. Foram desenhadas diferentes modificações para a lacase de modo a incrementar a sua performance, que incluíram a PEGilação e/ou imobilização. Ao mesmo tempo foram realizadas simulações de modelação molecular de forma a prever a geometria e as modificações estruturais da enzima. Durante o processo de polimerização, foram estudados três tipos de reatores com distintos níveis de energia associados. Os polímeros obtidos foram caracterizados e aplicados em diferentes substratos de forma a obter tecidos multi-funcionais. Este estudo visa fornecer conhecimento fundamental sobre a síntese de polímeros pela lacase promovendo ao mesmo tempo suporte teórico e técnico para o estabelecimento de sistemas catalíticos eficientes. Primeiramente, a polimerização do catecol foi feita usando a lacase como catalisador. O polietilenoglicol (PEG) foi usado na modificação não covalente e covalente da lacase sendo a sua performance posteriormente avaliada. A performance catalítica das diferentes lacases PEGiladas foi comparada em fase aquosa e em gel. Os resultados demonstram que a lacase modificada, quer de forma não-covalente ou covalente (lacase PEGilada), é capaz de promover a polimerização do catecol, aumentando quer o grau de conversão quer o grau de polimerização. No entanto, estes eventos foram unicamente detetados em fase aquosa. Os estudos de modelação molecular demonstram que a presença do PEG desacelera a desativação da enzima em fase aquosa. Posteriormente, foi efetuada a imobilização da lacase nativa e da lacase PEGilada em resina epoxy, usando polietilenoglicol como agente de ligação entre a enzima e o suporte. A performance de polimerização em fase aquosa das enzimas imobilizadas foi explorada e a estrutura dos polímeros obtidos foi proposta. Depois de imobilizada, o tempo de meia-vida da lacase foi incrementado assim como a sua estabilidade. A análise de MALDI-TOF MS revelou um aumento do grau de polimerização quando as enzimas imobilizadas, epoxy-lacase nativa, epoxy-PEGilada e epoxy-PEG-lacase, foram usadas como catalisador. Posteriormente, foram estudados diferentes reatores para a polimerização do catecol, nomeadamente banho termostatizado com agitação orbital, banho de ultrassons e homogeneizador de alta pressão. A atividade e estabilidade da lacase nesses reatores foram primeiramente avaliadas assim como o mecanismo de síntese do catecol em presença de ambientes de elevada energia. Os polímeros obtidos foram caracterizados sendo proposta a sua estrutura final. Os resultados demonstraram que ambientes de maior energia fornecem uma maior interação entre a enzima e o substrato, incrementando assim o rendimento de polimerização. O grau de conversão aquando da utilização quer do banho de ultrassons quer do homogeneizador de alta-pressão foi superior ao obtido quando a polimerização foi efetuada no banho termostatizado com agitação orbital. A lacase sob efeito de ultrassons ou do homogeneizador de alta pressão revelou menor estabilidade que quando sob o efeito do banho termostatizado. No entanto, dado que a polimerização ocorre antes dessa inativação, os ambientes de maior energia não provocam efeito negativo sobre a síntese do polímero. A performance catalítica das lacases, nativa, PEGilada e epóxi-PEGilada, foi avaliada quando sob o efeito do homogeneizador de alta pressão. Foi comparada a atividade e estabilidade assim como os correspondentes polímeros produzidos. As formas da lacase, PEGilada e Epoxi-PEGilada, revelaram a maior atividade catalítica assim como maior estabilidade. Em seguida, o catecol e a p-fenilenodiamina foram polimerizados no homogeneizador de alta-pressão pela lacase usando sacos feitos de poliéster (PET), algodão e lã que serviram de recipientes da enzima. A funcionalização dos tecidos foi conseguida através da polimerização in situ dos substratos aromáticos sobre os tecidos contendo a enzima. Ambos os polímeros, poli(catecol) e poli(p-fenilenodiamina), apresentam aceitável estabilidade dimensional e resistência à degradação térmica, assim como capacidade de eliminação de radicais livres. Foram obtidos polímeros com cor com capacidade de coloração dos tecidos usados. As observações efetuadas por microscópio eletrónico de varredura mostram uma distribuição uniforme do polímero na superfície dos tecidos de poliéster, algodão e lã, tendo os mesmos revelado alguma resistência à lavagem, à luz solar e à fricção. Os tecidos após funcionalização com poli(p-fenilenodiamina) revelaram condutividade elétrica. Ambos os polímeros produzidos foram capazes de conferir capacidade antimicrobiana aos tecidos com eles funcionalizados contra bactérias Gram positiva (S. aureus) e Gram negativa (E.coli). Ensaios de toxicidade efetuados nos tecidos funcionalizados revelaram que a difusão dos polímeros, depende da porosidade dos tecidos, e afeta diretamente a viabilidade celular, que pode ser aumentada ajustando a concentração do polímero. Este projeto visa o estudo do impacto da modificação da enzima e das condições de processamentos na estrutura dos produtos de reação com funções múltiplas, tendo ao mesmo tempo o objetivo de estabelecer um sistema reacional de catálise de uma panóplia de compostos fenólicos pela lacase. Os oligomeros e/ou polímeros produzidos poderão ter diferente aplicações em áreas como têxtil, médica, entre outras.
The research presented in this document was done in the Bioprocess and Bionanotechnology Research Group, Centre of Biological Engineering, University of Minho, Braga, Portugal. Part of this work was funded by the Portuguese Foundation for Science and Technology (FCT) through the grant CEB-BI-24-2017 and CEB-BI-06-2018 (strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01- 0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte). Part of this work was also funded by the Chinese Government Scholarship under China Scholarship Council (No. 201606790036), Chinese Foundation Key projects of governmental cooperation in international scientific and technological innovation (No. 2016 YFE0115700).

Chemical conjugation of a biomolecule, such as a peptide or a nucleic acid, with a functional molecule, for example a drug or a fluorophore, yields a bioconjugate, a polyfunctional entity that combines the properties of its components to elicit a biological effect. This kind of assemblies have found many successful applications, allowing the precise imaging of biological processes and delivering innovative treatments to the clinic, especially in oncology. The success of this class of constructs is determined by their ability to deliver the payload of choice to specific targets inside a biológical environment thanks to the targeting ability of the biomolecule. In order to have a functional conjugate, the payload must be linked to the targeting unit without altering the structure and function of the latter, preserving its ability to interact with the biological target. As a consequence, bioconjugation reactions must be performed in aqueous environment at physiological pH, avoiding strong reagents that might compromise the integrity of the biomolecule. Moreover, this ligation needs to be stable in physiological conditions while allowing the release of the payload in response to a stimulus that characterizes the biological target (pH, oxidative stress, enzymes). Boronic acids are a class of biocompatible reagents with the ability to form reversible covalent ligations in water, leading to their successful application in the development of stimulus-responsive bioconjugates. In this thesis, the 3-hydroxy quinolinone (3HQ) scaffold was studied as potential ligand for boronic acids in bioconjugation conditions, with the objective of using it as a functional unit for the reversible ligation of boronic acids to proteins. This scaffold was optimized for the desired purpose, yielding a structure that was successfully applied for the construction of a non-internalizing fluorescent bioconjugate. The resulting conjugate was tested in vitro, confirming its ability to deliver a borylated fluorescent probe to HT-29 cancer cells by targeting the 67 Laminin receptor. Additionally, the 3HQ motif was explored as multivalent modulator of human phenylalanine hydroxylase (hPAH), a protein whose mutation is the cause of phenylketonuria (PKU). In this study, various assays were performed to assess the ability of 3 series of derivatives to stabilyze the structure of hPAH and to increase its catalytic activity. Finally, the best performing compounds from this study were docked onto the structure of hPAH to gain additional insight on their activity.

One of the most important challenges in chemistry is the creation of new catalysts. Nature excels at this: constructed from biologically available elements, enzymes are versatile catalysts which adapt quickly to changing environments in order to sustain life. The combination of adaptable proteins with abiological reagents from synthetic chemistry affords a new direction for catalyst development. This thesis describes new enzymes, derived from a cytochrome P450 monooxygenase, which catalyze nitrogen and carbon atom transfer reactions to olefins and carbon−hydrogen bonds. Chapter 1 introduces directed evolution, a strategy for the laboratory optimization of proteins, in the context of improving metalloproteins for their native catalysis or for new reactions. Chapter 2 details the development of an enzyme-catalyzed transformation of olefins to aziridines, a valuable motif which is both present in bioactive molecules and used as a versatile building block for synthesis. This study establishes that when provided the appropriate reagents (e.g. styrenes and tosyl azide), heme proteins can adopt a nitrene transfer catalytic cycle to form aziridine products and that the turnover and selectivity of the catalyst can be optimized through mutation of its amino acid sequence. The activity of heme protein catalysts is extended to the functionalization of sp3 hybridized C−H bonds for carbon–nitrogen and carbon–carbon bond formation through nitrene and carbene insertion respectively (Chapters 3 and 4). With the exception of C−H oxygenation chemistry, iron complexes are under-utilized for sp3 C−H functionalization reactions, despite iron being readily available and non-toxic. Combining previously engineered heme proteins with suitable substrates led to initial reaction discovery. Directed evolution of these enzymes significantly improved their C−H functionalization activity (by 140-fold in Chapter 4). Characterization of evolved enzymes, including the attainment of an X-ray crystal structure (Chapter 3) and substrate scope studies (Chapters 3 and 4), were pursued. In sum, the thesis work addresses both the biological question of expanding the catalytic capabilities of existing enzymes through mutation and expands the chemistry of iron-porphyrin catalysts.