Thèses sur le sujet « Protein material »

Under det här kandidatexamensarbetet har protein-nanofibers påverkan på material undersökts genom att jämföra fibrillerade filmer med ofibrillerade. Sojaproteinisolat fibrillerades under förhållandena pH 2 och 85 ◦C under minst ett dygn och de syntetiserade nanofibrerna analyserades med Thioflavin T (ThT) fluorescens och atomkraftsmikroskopi (AFM). Spektra från analysmetoden ThT fluorescens indikerade på förekomsten av β-flak och analyserna med AFM visade på att fibrerna hade en morfologi som är karakteristisk för protein-nanofibrer. Resultaten antyder att de parametrar som påverkar morfologin hos fibrerna är fibrilleringstid och typ av protein. De gjutna filmerna från fibrillära respektive ofibrillära proteiner var sammanhängande bortsett från vissa sprickor. Värdena på E-modulen från AFM visade att det fibrillerade materialet var mer heterogent än det ofibrillerade. Filmer med sammanhängande yta erhölls vid tillsats av det mjukgörande additivet glycerol. Slutligen, material av både fibrillär och ofibrillär form kan framställas, däremot krävs vidare forskning för att optimera materialens egenskaper.
During this bachelor thesis project, the impact of protein nanofibers on materials has been analysed by comparing films made from fibrillar and non-fibrillar protein. Fibrillation of soy protein isolate was performed during at least 24 hours at pH 2 and a temperature of 85 ◦C. Analysis of the nanofibers was made with Thioflavin T (ThT) fluorescence and atomic force microscopy (AFM). The spectra from ThT Fluorescens indicated the presence of β-sheets and AFM confirmed that the fibrils had a morphology that is characteristic of protein nanofibers. The results indicated that heating time and protein type were the parameters which had the largest impact on the morphology of the fibrils. The synthesised films from both fibrillar and non-fibrillar protein were coherent with exception of some cracks. The elastic modulus from AFM indicated that the fibrillar film was more heterogeneous compared to the non-fibrillar film. To attain coherent films, the plasticising agent glycerol was added. To summarise, both fibrillar as well as non-fibrillar materials were successfully synthesised, however, further research is necessary to optimise the properties of the material.

The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption.

Five different ceramic biomaterials: alumina (Al₂O₃), zirconia (ZrO₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and two glass-ceramics, AP40 (SiO₂-CaO-Na₂O-P₂O₅-MgO-K₂O-CaF₂) and RKKP (AP40 with Ta₂O₃-La₂O₃), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an in vivo setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration in vivo.

Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α₂-HS-glycoprotein and α₁-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations.

The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α₂-HS-glycoprotein and α₁-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material.

This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.

Esta tesis aborda la actividad biológica de la fibronectina (FN) como proteína de interfase en la interacción célula-material. La tesis investiga la respuesta de la proteína, en términos de cantidad adsorbida y conformación, ante diferentes propiedades físico-químicas del material. Además, se correlaciona la respuesta celular temprana y la funcionalidad celular con el estado de la proteína adsorbida sobre el material. Para ello se prepararon diferentes series de materiales con propiedades físico-químicas controladas. La distribución de FN sobre las diferentes superficies se caracterizó mediante el uso de la microscopía de fuerza atómica (AFM) y la densidad superficial adsorbida fue cuantificada mediante técnicas de marcado radioactivo y western blot. La respuesta celular se evaluó en términos de la adhesión inicial a las superficies, así como los procesos posteriores de diferenciación, proliferación, reorganización y producción de matriz extracelular. Se investigó el efecto de la nanotopografía en la adsorción de la FN y el comportamiento celular sobre una serie de topografías controladas en la escala nanométrica, obtenidas mediante el spin casting de soluciones de ácido poli(L-láctico)/poliestireno (PLLA/PS) de distintas concentraciones. La migración del PLLA hacia la superficie del film durante el proceso de spin coating proporciona superficies de PLLA con nanopicos de diferentes tamaños (14, 29 y 45 nm). El tamaño de la nanoestrutura afecta a la densidad de FN adsorbida, siendo mayor en la superficie de menor nanotopografía. En cuanto a la respuesta celular inicial, se observan adhesiones focales más desarrolladas y mejor reorganización celular de la capa de FN adsorbida en las superficies de mayor topografía (29 and 45 nm), lo que resulta en una mayor producción y organización de nueva matriz. Por otra parte se empleó una familia de materiales con sutiles variaciones en la composición química: polímeros acrílicos (polimetil, etil y butil acrilato -PMA, PEA y P
González García, C. (2012). BIOLOGICAL ACTIVITY OF FIBRONECTIN AT THE CELL-MATERIAL INTERFACE [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17701
Palancia

Activated platelets can recruit monocytes into thrombi through the P-selectin*PSGL-1 interaction. Cross-talk between the cells may regulate monocyte gene and protein expression. A previous genome-wide transcriptomic study identified >3000 genes upregulated in monocytes in response to activated platelets, including a number of antithrombotic genes; in particular tfpi and procr. This thesis aimed to establish the mechanisms by which platelets regulate these genes, and if this was through the nuclear receptor PPARγ. Using the same experimental model as in previous studies, in which platelets were activated in whole blood with the platelet-specific GPVI agonist CRP-XL, monocyte tfpi and procr expression was confirmed, shown to be affected by inhibitors of COX-1 (aspirin) and 12-LOX (esculetin and baicalein), and regulated through soluble factors released from platelets, which were shown to be oxylipins for tfpi and proteins ~10kDa for procr. Expression of pparγ was also increased and largely regulated through direct platelet-monocyte contact, although oxylipins potentiated expression. An associated increase in protein expression was partially confirmed for all three genes. Mass spectrometry (MS) of platelet-derived releasate measured 386 proteins and identified platelet factor 4 (PF4) and RANTES (CCL5) as likely candidates for procr regulation. Expression was attenuated with releasate incubated with heparin agarose (HA) but this was not rescued with exogenous PF4. LC-MS/MS measured no change in PF4 levels in the releasate incubated with HA but complete removal of RANTES. MS identified 10 oxylipins released from platelets (AA, 8-, 9-, 11-, 12-, 15-HETE, 9-, 13-HODE, PGD/E2 and TxA2) of which four (11-, 15-HETE, 9-, 13-HODE) were significantly, and TxA2 and PGD/E2 completely reduced by aspirin, and all but AA and 9-HETE reduced with 12-LOX inhibitors. These oxylipins included known PPARγ agonists. Incubation with 15d-PGJ2 and rosiglitazone confirmed potentiation of tfpi expression in monocytes. Using a transactivation assay 12- and 15-HETE were shown to activate PPARγ expression in vitro, while X-ray crystallography indicated, for the first time, interaction of both with PPARγ. These results are the first to show regulation of antithrombotic genes in monocytes by factors released from platelets. It is the first to profile oxylipins released from GPVI-activated platelets and identify PPARγ as a regulator of tfpi, and RANTES as a potential regulator of procr expression in monocytes. The observation that aspirin attenuates expression of both these genes raises issues with its use in the treatment of cardiovascular disease, for which it is relied on heavily.

Supramolecular proteins that assemble into cage like architectures have been used for nano-material synthesis and as a scaffold for metal binding. Material synthesis can be performed by exploiting the cage-like properties of these nano-containers and relying on the electrostatically distinct interior environment that drive mineral encapsulation. Ferritin and ferritin like proteins can be used as size constrained reaction vessels that encapsulate materials that have sizes that are determined by the internal dimensions of the protein cage. These range from 5 nm for the ferritin like protein from Listeria innocua to 24 nm for the interior of an engineered plant virus (Cowpea chlorotic mottle virus). Inorganic materials synthesized within these constrained reaction volumes are monodisperse in size. The crystallinity and phase of material prepared is determined by the reaction conditions, which are mild compared to other preparative methods. The metal binding affinity of certain viral protein cages allows the study of the role that metals play in such processes as viral assembly and infection as well as transmission. If paramagnetic metal ions are bound to the viral protein cage, the biological scaffold has potential use as an MRI contrast agent. Here multiple protein cage platforms are discussed with an emphasis on engineering non-native functionality to many of the protein cages. Engineering nonnative function to protein cages involves both genetic and chemical modification for the purpose of increasing stability and changing electrostatic interactions. Together these modifications serve to reinforce the versatility of protein cage architectures for both mineral synthesis and metal binding.

This thesis ventures with the extracellular matrix protein (ECM) fibronectin (FN) as an interface protein in the interaction between cells and materials to design microenvironment for future use in tissue engineering. It is studied the FN adsorption and conformations, cell behaviour to different FN conformation, cell adhesion, reorganisation and remodelling of FN at the material interface, the role of growth factors (GF) and their interactions with components of the extracellular matrix (ECM), the immunology cell response, and the stem cell fate influenced by the extrinsic signals coming from the engineered microenvironments using ECM's proteins. To investigate the FN response, in terms of adsorbed amount and conformation to different chemical properties of the material, model surfaces were used. Self assembled monolayers (SAM) with different percentages of two different chemical groups were used: CH3 and OH. FN adsorption, initial cell adhesion and signalling (focal adhesions, integrin expression and phosphorylation of FAK) is related with the reorganisation and secretion of FN and matrix degradation. It is shown that matrix degradation at the cell material interface depends on surface chemistry in metalloproteinase-dependent way. A direct relationship between FN activity at the cell-material interface and metalloproteinase 9 (MMP9) expression was found, being the product of a sequence of events that include integrin expression, focal adhesion formation, matrix reorganisation and focal adhesion kinase (FAK) phosphorylation. Two different materials with subtle variations in their chemical composition were employed as a drastically different FN conformation: from a globular conformation on PMA (poly (methyl acrylate)) to the formation of a well-interconnected FN network (similar to the FN physiological fibrillar network) triggered by PEA (poly (ethyl acrylate)). The formation of focal adhesions (vinculin), FAK expression and phosphorylation, specific integrin binding, protein and gene expression for ¿5 and ¿v was studied, seeking to correlate cell adhesion with matrix degradation. It is demonstrated that the material-driven FN fibrillogenesis on PEA triggers proteolytic activity: MMP activity is higher as a compensatory mechanism to the inability of cells to reorganise this FN network. Looking into the role of protein-material interactions and stem cell fate, and with the knowledge on PEA, we engineer different synergistic microenvironments to direct cell and stem cell fate. FN has a growth factor (GF) binding domain on its molecule (FNIII12-14) and has been demonstrated to produce a synergistic response when occurs at the same time the recognition of the cell binding domain (FNIII9-10). It is demonstrated that this domain is available on the FN coated PEA, and exploiting these interactions between PEA, FN and GF, it is developed a microenvironment to control cell behaviour and tissue repair. It is studied the BMP2 binding and presentation, the effect of BMP2 presentation on MSC proliferation and differentiation. These systems allow not only enhanced activity of GF compared to soluble administration, but also reduce GF doses, improving safety and cost effectiveness. Finally, the immunological reaction of the microenvironment developed is studied using dendritic cells, beside the conformational structure of ECM protein importance in DC integrin-based activation it is studied, helping to establish the field of adhesion-based modulation of DC as a general mechanism that has previously not been defined. The microenvironment didn't induce any maturation in DC, while different FN conformation shows differences in DC morphology and citokine level production (IL-10 and IL-12).
En esta tesis se estudia la interacción de una proteina de la matriz extracelular, fibronectina (FN) como interfase en la interacción entre células y materiales, para diseñar microambientes con el propósito de ser usados en el futuro en ingeniería tisular. Se estudia la adsorción y conformación de FN y la relación con el diferente comportamiento celular: la adhesión celular, la reorganización y remodelado de la FN en la interfase célula-material, el papel que juegan los factores de crecimiento y sus interacciones con los componentes de la matriz extracelular, la respuesta immunológica y el destino celular de células madre influenciadas por las señales extrínsecas provenientes de microambientes elaborados a partir de proteínas de la matriz extracelular. Con el objetivo de investigar la respuesta a la FN en términos de conformación y cantidad absorbida a diferentes propiedades químicas del material, se usaron materiales modelo: monocapas autoensambladas (self-assembled monolayers, SAM). Las químicas estudiadas fueron CH3 and OH. La adsorption de FN, adhesion y señalización (adhesiones focales, expresión de interinas y fosforilación de quinasas de adhesiones focales (FAK)) se estudiaron en relación a la reorganización y secreción de FN y degradación de la matriz extracelular. Se demuestra que la degradación de la matriz extracelular en la interfase célula-material depende de la química de la superficie, a través de las metaloproteinasas. Se ha descubierto una relación directa entre la actividad de la FN que se encuentra en el material y la expresión de metaloproteinasa 9 (MMP9), a través de la expresión de integrinas, formación de adhesiones focales, reorganización de la matriz extracelular y fosforilación de FAK En el siguiente capítulo se emplean materiales poliméricos con una sutil diferencia en la composición química, provocando una diferencia drástica en la conformación de la FN: se pasa de una conformación globular en PMA (polimetil acrilato) a una conformación en forma de red interconectada en PEA (polietil acrilato). Con el propósito de relacionar la adhesión celular con la degradación de la matriz extracelular, se estudia la formación de adhesiones focales (vinculina), la expresión y fosforilación de FAK, la unión específica de integrinas y la expresión de las integrinas ¿5 and ¿v. Se demuestra que la formación de una red de FN sobre PEA induce la actividad proteolítica: la actividad de las MMPs es mayor, actuando como mecanismo compensatorio a la incapacidad de reorganización de la red de FN. Haciendo uso de la conformación de la FN sobre PEA, se estudiaron las interacciones entre la proteína-material y el destino celular de células madres. La FN posee un dominio de unión de factores de crecimiento (FNIII12-14) y se ha demostrado que se produce una respuesta sinérgica cuando el reconocimiento ocurre junto con el dominio de unión celular (FNIII9-10). En esta tesis se demuestra que el dominio de unión de factores de crecimiento está disponible en la conformación que adquiere sobre PEA y se diseñan microambientes para controlar el comportamiento celular y regeneración de tejido. Se estudia la unión y presentación de BMP2 y su efecto en la diferenciación de células madre mesenquimales. Los microambientes desarrollados, ademas de mejorar la actividad de los factores de crecimiento comparado con la administración soluble, también reduce la cantidad de factores de crecimiento que se tendría que administrar, mejorando la seguridad y efectividad. Finalmente se estudió la reacción inmunológica a los microambientes desarrollados usando células dendríticas, estudiando además la influencia de la estructura de la conformación de las proteínas en la activación de las células dendríticas a través de las integrinas. Los microambientes no indujeron ninguna maduración de células dendríticas, mientras que la conformación de la FN muestra control
En aquesta tesi s'estudia la interacció entre una proteïna de la matriu extracel.lular, fibronectina (FN) com interfase en la interaccio entre cèl·lules i materials, per a dissenyar microambients amb el propòsit d'utilitzar-se al futur en enginyeria tissular. S'estudia l'adsorció i conformació de la FN i la relació amb el diferent comportament cel·lular: l'adhesió cel·lular, la reorganització i remodelat de la FN a la interfase cèl·lula-material, el paper que juguen els factors de creixement i les seus interaccions amb els components de la matriu extracel·lular, la resposta immunològica i el destí cel·lular de cèl·lules mare influenciades pels senyals extrínseques provinents de microambients elaborats a partir de proteïnes de la matriu extracel·lular. Amb l'objectiu d'investigar la respostar a la FN en termes de conformació i quantitat absorbida a diferents propietats químiques del material, s'utilitzaren materials model: monocapes autoacoblades (self-assembled monolayers, SAM). Les químiques estudiades van ser CH3 and OH. L'absorció de FN, adhesió i senyalització (adhesions focals, expressió d'integrines i fosforilació de quinases d'adhesions focals (FAK)) es van estudiar en relació a al reorganització i secreció de la FN i degradació de la matriu extracel·lular. Es demostra que la degradació de la matriu extracelular en la interfase cèl·lula-material depèn de la química de la superficie, a través de les metal·loproteïnases. S'ha descobert una relació directa entra l'activitat de la FN que es troba en el material i l'expressió de metaloproteinasa 9, a través de l'expressió d'integrines, formació d'adhesions focals, reorganització de la matriu extracel·lular i fosforilació de FAK. Al següent capítol es fan servir materials polimèrics amb una subtil diferència en la composició química, provocant una diferència dràstica en la conformació de la FN: es passa d'una conformació globular en PMA (polimetil acrilat) a una conformació en forma de xarxa interconnectada en PEA (polietil acrilat). Amb el propòsit de relacionar l'adhesió cel·lular amb la degradació de la matriu extracel·lular, s'estudia la formació d'adhesions focals (vinculina), l'expressió i fosforilació de FAK, la unió específica d'integrines i l'expressió de les integrines ¿5 and ¿v. Es demostra que la formació d'una xarxa de FN sobre PEA indueix l'activitat proteolítica: l'activitat de les MMPs és més gran, actuant com a mecanisme compensatori a la incapacitat de reorganització de la xarxa de FN. Fent ús de la conformació de la FN sobre PEA, es van estudiar les interaccions entre la proteïna-material i el destí cel·lular de cèl·lules mares. La FN posseeix un domini d'unió de factors de creixement (FNIII12-14) i s'ha demostrat que es produeix una resposta sinèrgica quan el reconeixement ocurreix juntament amb el domini d'unió cel·lular (FNIII9- 10). En aquesta tesi es demostra que el domini d'unió de factors de creixement està disponible a la conformació que adquireix sobre PEA i es dissenyen microambients per controlar el comportament cel·lular i regeneració de teixit. S'estudia la unió i presentació de BMP2 i el seu efecte en la diferenciació de cèl·lules mare mesenquimals. Els microambientes desenvolupats, a més de millorar l'activitat dels factors de creixement comparat amb l'administració soluble, també redueix la quantitat de factors de creixement que s'hauria d'administrar, millorant la seguretat i efectivitat. Finalment es va estudiar la reacció immunològica als microambients desenvolupats usant cèl·lules dendrítiques, estudiant a més la influència de l'estructura de la conformació de les proteïnes en l'activació de les cèl·lules dendrítiques a través de les integrines. Els microambients no van induir cap maduració de cèl·lules dendrítiques, mentre que la conformació de la FN mostra controlar la morfologia de les cèl·lules dendrítiques i
Llopis Hernández, V. (2017). Material-driven fibronectin fibrillogenesis to engineer cell function [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90412
TESIS

Access to fresh water is a human right, yet more than 780 million people, especially in rural areas, rely on unimproved sources and the need for finding ways of treating water is crucial. Although the use of natural coagulant protein in drinking water treatment has been discussed for a long time, the method is still not in practice, probably due to availability of material and limited knowledge. In this study, about hundred different crude extracts made from plant materials found in Southern India were screened for coagulation activity. Extracts of three Brassica species (Mustard, Cabbage and Cauliflower) were showing activity comparable to that of Moringa oleifera and were further investigated. Their protein content and profile were compared against each other and with coagulant protein from Moringa. Mustard (large) and Moringa seed proteins were also studied for their effect against clinically isolated bacterial strains. The protein profiles of Brassica extract showed predominant bands around 9kDa and 6.5kDa by SDS-PAGE. The peptide sequence analysis of Mustard large identified the 6.5kDa protein as Moringa coagulant protein (MO2.1) and the 9kDa protein band as seed storage protein napin3. Of thirteen clinical strains analysed, Moringa and Mustard large were proven effective in either aggregation activity or growth kinetic method or both in all thirteen and nine strains respectively. To my knowledge this is the first report on the presence of coagulant protein in Brassica seeds. Owing to the promising results Brassica species could possibly be used as a substitute to Moringa coagulating agent and chemicals in drinking water treatment.

QC 20121214

When we stretch and contract a rubber band a hundred times, we expect the rubber band to fail. Yet our heart stretches and contracts the same amount every two minutes, and does not fail. Why is that? What causes the significantly higher elasticity of certain molecules and the rigidity of others? Equally importantly, can we use this information to design materials for precise mechanical tasks? It is the aim of this dissertation to illuminate key aspects of the answer to these questions, while detailing the work that remains to be done. In this dissertation, particular emphasis is placed on the nanoscale properties of elastomeric proteins. By better understanding the fundamental characteristics of these proteins at the nanoscale, we can better design synthetic rubbers to provide the same desired mechanical properties.
Ph. D.

Activated sludge influent and effluent from five municipal wastewater treatment plants were analyzed to further elucidate the roles of aluminum, iron, and the monovalent to divalent cation ratio (M/D) on the influent and effluent characteristics of the systems. The size distribution of organic nitrogen, organic carbon, protein, humic acid, and polysaccharide was examined with respect to the concentration of cations in the activated sludge influent. It was found that the majority of organic nitrogen, organic carbon, protein and polysaccharides were found in material larger than 0.45μm in activated sludge influent. Humic acids were mostly found in material smaller than 0.45μm. Protein was the largest contributor to organic nitrogen and humic acids were the largest contributor to organic carbon. Using 0.45μm as a division between particulate and soluble material, the ratio of soluble to particulate material in activated sludge influent was found to be negatively correlated with the ratio of iron to aluminum. In activated sludge effluent, the majority of the organic nitrogen and protein was found in material larger than 0.45μm, while the majority of the organic carbon, humic acid, and polysaccharide were found in material smaller than 30kDa. Influent aluminum concentration had no observable effect on the concentration or distribution of organic nitrogen or organic carbon. Influent iron appeared to play a role in the flocculation of organic nitrogen and protein containing material between 0.45μm and 1kDa in size. A high monovalent to divalent cation ratio appeared to play a role in deflocculating organic nitrogen containing material larger than 1.5μm and increased the concentration of TOC smaller than 1kDa and the total polysaccharide concentration. Tertiary depth filtration removed all organic nitrogen and protein in material larger than 0.45μm, but a poor job of removing organic carbon from and an inconsistent job of removing polysaccharide from effluent Eight lab-scale activated sludge reactors were also run, but the data was not consistent enough for analysis and comparison with the municipal wastewater treatment plants. This thesis contains a series of four papers that each deal with a different aspect of the role of cations on activated sludge influent and effluent. The first paper focuses on activated sludge influent characteristics, the second on effluent organic nitrogen and organic carbon, the third on effluent EPS, and the last on the lab-scale reactors. The papers were divided in this way because of the unique analytical obstacles that were encountered with each set of data.
Master of Science

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 136-146).
From the fabrication of fine chemicals, to the increasing attainability of a non-petrochemical based energy infrastructure, catalysts play an important role in meeting the increasing energy and consumable demands of today without compromising the global health of tomorrow. Development of these catalysts relies on the fundamental understanding of the effects individual catalyst properties have on catalytic function. Unfortunately, control, and therefore deconvolution of individual parameter effects, can be quite challenging. Due to the nanoscale formfactor and wide range of available surface chemistries, biological catalyst fabrication affords one solution to this challenge. To this end, this work details the processing of M13 bacteriophage as a synthetic toolbox to modulate key catalyst parameters to elucidate the relationship between catalyst structure and performance. With respect to electrocatalysis, a biotemplating method for the development of tunable 3D nanofoams is detailed. Viral templates were rationally assembled into a variety of genetically programmable architectures and subsequently templated into a variety of material compositions. Subsequently, this synthetic method was employed to examine the effects of nanostructure on electro-catalytic activity. Next, nanoparticle driven heterogeneous catalysis was targeted. Nanoparticle-protein binding affinities were leveraged to explore the relationship between nanoparticles and their supports to identify a selective, base free alcohol oxidation catalyst. Finally, the surface proteins of the M13 virus were modified to mirror homogeneous copper-ligand chemistries. These viruses displayed binding pocket free copper complexation and catalytic efficacy in addition to recyclability and solvent robustness. Subsequently, the multiple functional handles of the viron were utilized to create catalytic ensembles of varying ratios. Single and dendrimeric TEMPO (4-Carboxy-2,2,6,6-tetramethylpiperidine 1-oxyl) were chemically conjugated to the surface of several catalytically active phage clones further tailoring catalytic function. Taken together, these studies provide strong evidence of the utility of biologically fabricated materials for catalytic design.
by Jacqueline Ohmura.
Ph. D.

学位授与年月日: 2007-07-23 ; 学位の種類: 新制・論文博士 ; 学位記番号: 論工博第3968号
GENERAL INTRODUCTION: Bone reconstruction is a clinically important procedure to treat bone defects and has been widely tried by different methods. Basically, bone has the inherent ability to spontaneously repair itself for the bone fracture of small size. However, such a self-repairing cannot always be expected for large-size defects that are caused by trauma, tumor resection, spinal arthodesis, and congenital abnormalities. This situation often happens clinically and the therapeutic demand has been being increased recently [1]. Autograft, which is considered to be a gold standard as bone substitutes, is applied to the defect site because it provides a suitable environment for cell attachment, proliferation, and differentiation for bone regeneration [2]. However, it has several disadvantages, such as the limited donor supply, potential complications with chronic pain at the donor sites [3, 4]. On the other hand, allograft is being performed clinically [2], but the rate of graft integration into the surrounding natural bone is lower than that of autograft. In addition, it is necessary for the allograft to consider a risk of disease transmission and postoperative complications due to the tissue rejection [4, 5]. Therefore, under these circumstances, as the substitute for the bone grafts, the biomaterials of metals and ceramics have been investigated and developed. Although the above problems may be cleared, they have other disadvantages, such as the lack of biodegradability under physiological conditions and the limited processability [6]. Especially, metals show poor integration property to the bone tissue at the implantation site compared with the autograft and allograft although they provide mechanical support [7]. Different from artificial biomaterials, one of the important advantages for the bone graft is to positively accelerate osteoconduction and osteoinduction. As one trial to tackle and improve the points to be resolved, bone tissue engineering has been attracted much attention as a new therapeutic technology [8-11]. The basic idea is to provide key cells the local environment suitable to promote their proliferation and differentiation for the induction of tissue regeneration. (...)　In summary, this thesis describes the feasibility of gelatin-based scaffolds in the controlled release carrier of BMP-2 and the three-dimensional matrix of MSC for osteogenic differentiation. It is concluded that this material design of scaffold is promising to effectively induce bone regeneration based on tissue engineering.
京都大学
0048
新制・論文博士
博士(工学)
乙第12103号
論工博第3968号
新制||工||1418(附属図書館)
UT51-2007-M983
(主査)教授 田畑 泰彦, 教授 岩田 博夫, 教授 木村 俊作
学位規則第4条第2項該当
Doctor of Engineering
Kyoto University
DFAM

Large White turkey breeder hens were used to examine the effect of duration of feed restriction, prebreeder protein content, and nesting material on subsequent growth and reproductive performance. Day old poults were raised following standard commercial practices with feed and water for ad libitum consumption until 6 wk of age (WOA). At this time, hens were equally divided among six grower feeding regimens. The treatments were as follows: a) a control group fed standard commercial diets for ad libitum consumption (CON); b) a second control group like (a) but fed plain white oats from 19 through 26 wk (OATS). In contrast, the 4 remaining treatment groups were feed restricted beginning at 6 WOA to achieve body weights 45% less than the full-fed CON at 16 WOA. Birds were kept at this level of restriction until either 17.0 (R17.0), 18.3 (R18.3), 19.6 (R19.6), or 20.9 (R20.9) WOA. Thereafter, feed allowance was gradually increased to achieve a predetermined minimum target BW of 10.8 kg at photostimulation. An additional prebreeder protein treatment was superimposed from 27 to 31 WOA. Treatments reduced BW but none of the four quantitatively restricted groups achieved the target BW of 10.8 kg at photostimulation. Feed restriction reduced feed consumption and improved feed conversion. There were no differences in flock uniformity, sexual maturity, mortality, body composition at photostimulation, and total egg production. The R18.3 treatment achieved the highest peak production. The quantitative restriction treatments exhibited low laying persistency. There were no differences in number of large yellow follicles, egg weight, fertility, or hatchability, but poult weight was reduced in the R18.3 treatment. Dietary protein influenced the proportions of multiple follicle sets and percentage misshaped eggs.Three nesting materials were compared and were as follows: 1) all nests filled with shavings (S), 2) all nests filled with paper chips (P), and 3) two nests filled with shavings and two nests with paper chips (S/P). There were significant differences in percentage floor and broken eggs. Nesting materials did not affect total egg production, fertility, or hatchability. The data suggest, if restriction is too severe and is continued too near to the time of conventional photostimulation, BW recovery and egg production will be depressed. Oat feeding was the easiest treatment to implement and resulted in equivalent reproductive performance. Dietary protein content may affect proportions of multiple follicle sets. Turkey breeder hens can and do distinguish between nesting materials and this may affect floor laying. Combinations of various types of nesting materials within the same breeder unit should be avoided. (Key words: turkey breeder hens, feed restriction, prebreeder protein, egg production, body composition, nesting material)
Ph. D.

The objective of this thesis was to investigate the role of hydroxyapatite and silicate-substituted hydroxyapatite synthetic bone graft substitute (SBG) material properties in modulating the processes of protein adsorption and desorption, and their combined role in the subsequent regulation of cell attachment, proliferation and differentiation on the surfaces of these materials in vitro. As a result of their purported role in promoting osteogenic behaviour in vivo the materials parameters selected for investigation were chemistry (stoichiometric hydroxyapatite (HA) versus 0.8wt% silicate-substituted hydroxyapatite (SA)) and strut porosity (20% versus 30% strut porosity). Cell attachment and response to different SBG was assessed to samples in the ‘as received’ condition as well as after a series of sequentially varied pre-treatments with solutions of phosphate buffered saline or cell culture media either unsupplemented or in combination with mixed serum proteins and/or Fibronectin (Fn). This enabled investigation of the effect of sample chemistry and strut porosity on mixed serum protein interactions and Fn adsorption under both competitive and non-competitive conditions, and the study of subsequent regulation of cell attachment and response as a consequence of pre-treatment. Results showed that serum protein interactions were key to modulation of cell response to chemistry, and there was evidence that for Fn this may be related to conformational changes in the adsorbed protein rather than its level of enrichment in the protein interlayer. In terms of the materials properties investigated strut porosity was found to be the most dominant factor in the regulation of cell response, where SBG with 30% strut porosity promoted human mesenchymal stem cell (hMSC) osteoblastic differentiation. Moreover hMSC response to SBG with 30% strut porosity seemed to be less sensitive to pre-treatment. In conclusion, the results of these experiments indicate that strut porosity more directly influences the cellular response to HA and SA BGS than chemistry in vitro. Moreover, the role that Fn and other serum proteins have in regulating this response is dependent on the physiological environment and BGS chemistry.

Atualmente, há um grande incentivo governamental para a produção de biodiesel a partir de óleo de mamona. O aumento na fabricação desse óleo irá aumentar a produção de torta de mamona, que tem grande potencial de emprego na tecnologia de material biodegradável e utilização na alimentação animal, se destoxificada. Os objetivos desta tese foram a caracterização química da torta de mamona, a extração de suas proteínas para desenvolvimento de material biodegradável, e a caracterização do resíduo sólido do processo de extração, visando seu uso na alimentação animal. A extração das proteínas da torta de mamona foi feita por solubilização em meio alcalino. Inicialmente, diversos parâmetros (velocidade de agitação, concentração da torta na solução extratora e temperatura de extração) foram testados no intuito de aumentar o rendimento de extração das proteínas, utilizando-se NaOH (pH = 9). Em seguida, diversos experimentos foram realizados para se avaliar os efeitos do pH (8-12) e/ou do tipo de agente alcalino (NaOH, KOH e Ca(OH)2) na extração das proteínas da torta de mamona, sempre à temperatura de 50°C, velocidade de agitação de 400rpm, e concentração da torta na solução extratora de 20%. Os extratos proteicos obtidos foram liofilizados (EPL), e os resíduos foram desidratados em estufa (40°C/24 horas). Análises para determinação da composição bromatológica, minerais, amido, compostos fenólicos totais, ácidos graxos, aminoácidos, fibra dietética, da microestrutura, atividade alergênica, eletroforese dimensional e identificação de ricina foram feitas na matéria prima, nas proteínas extraídas e nos resíduos. A composição de aminoácidos foi analisada no extrato proteico liofilizado (EPL) e nos resíduos somente nas amostras preparadas com NaOH. A composição bromatológica das matérias primas (semente, torta e farelo) mostrou elevados teores de óleo (43,6%) e fibras (29,5%) para as sementes; bem como altos teores de proteína (36-40%) e fibras (29-30%) para a torta e o farelo de mamona. A semente de mamona apresentou valores de minerais considerados razoáveis para oleaginosas, e a torta de mamona apresentou alta proporção de K; Ca e P, importantes para alimentação animal. Verificou-se alto teor de ácido ricinoléico (79-90%) nas matérias primas avaliadas. A torta de mamona apresentou altos teores de ácido glutâmico (15%), arginina (11%) e triptofano (9%), sendo que os teores dos outros aminoácidos variaram entre 2 e 7%. Os resultados da caracterização química dos EPL e dos resíduos foram dependentes principalmente do pH de extração das proteínas. Observou-se aumento da concentração de proteína (64-68%) e de cinzas (13-19%), nos EPL, em função do aumento do pH (10 - 12), independente do tipo de agente alcalinizante. Os resíduos obtidos da extração dos EPL apresentaram teores de proteína, cinzas e fibras variando entre 20 e 34%, 12 e 17% e 39 e 42%, respectivamente. Em relação ao perfil de minerais dos EPL, verificou-se aumento nas concentrações de Na e Mn com o aumento do pH para ambos agentes alcalinizantes. O perfil de minerais dos resíduos mostrou aumento significativo nos teores de Na e K com aumento do pH de extração, ajustado com NaOH e KOH, respectivamente. O teor de ácido ricinoléico foi menor no resíduo, e maior no EPL, obtidos em pH 12 ajustado com NaOH. A composição de aminoácidos dos resíduos sofreu efeito do pH apenas para o pH 12. Os aminoácidos presentes em maior concentração nos EPL e nos resíduos foram ácido glutâmico, triptofano e arginina. Os EPL e os resíduos obtidos da extração das proteínas apresentaram menor poder alergênico avaliado pelo teste de desgranulação de mastócitos obtidos do lavado peritoneal de ratos. O perfil eletroforético dos EPL e dos resíduos mostrou maior proporção de proteínas com massa molecular entre 29-36kDa, seguidos por proteínas de massa molecular ao redor de 20kDa e menor proporção de proteínas de massa molecular entre 45-66kDa. Não foi observada a presença da ricina nos resíduos obtidos em pH 12. Os resultados de todos os experimentos de extração das proteínas da torta de mamona permitiram escolher os melhores parâmetros de extração: temperatura = 50°C, velocidade de agitação = 400rpm, concentração da torta na solução extratora = 20%, pH = 12 e agente alcalinizante = NaOH. Em conclusão, o processo de extração de proteínas por solubilização alcalina permitiu a produção de concentrado proteico com características interessantes para a produção de material biodegradável (agricultura e biofilmes), e de resíduos ainda com alto teor de proteínas e de fibras, isento de ricina, podendo, portanto ser utilizado na alimentação animal.
Presently, there is large government incentive for biodiesel production from castor seed oil. The increase of this oil fabrication will raise the production of castor seed pulp, which has great potential for utilization for biodegradable material technology and for animal feed, if detoxified. The objectives of this research were the chemical characterization of the castor seed pulp, the extraction of the proteins for biodegradable material developing, and the characterization of the solid residue of the extraction process, aiming its use for animal feed. The protein extraction from the castor seed pulp was made through solubilization in alkaline medium. Initially, several parameters (stirring speed, pulp concentration in the extraction solution and extraction temperature) were tested aiming the optimization of the protein extraction yield, using NaOH (pH 9). Subsequently, several experiments were realized to evaluate the effects of pH (8-12) and/or of the type of alkaline agent (NaOH, KOH and CaOH) on the castor seed pulp protein extraction, always at 50 °C, 400rpm stirring speed, and 20 % pulp concentration in the extraction solution. The obtained protein extracts were lyophilized (EPL), and the residues were dehydrated in stove (40 °C/24 h). The raw material, the extracted proteins and the residues were analyzed for bromatologic composition, minerals, starch, total phenolic compounds, fatty acids, amino acids, dietetic fiber, microstructure, allergenic activity, dimensional electrophoresis and ricin identification. The amino acids composition was analyzed on the lyophilized extract (EPL) and in the residues of the samples prepared with NaOH. The bromatologic composition of the raw materials (seed, pulp and meal) showed high content of oil (43.6 %) and fiber (29.5 %) for the seeds; as well as high amounts of protein (36-40 %) and fiber (29-30 %) for the pulp and the castor seed meal. The castor seed had average mineral values for oil seeds, and the castor seed pulp showed high percentage of K, Ca and P, valuable for animal feed. The evaluated raw materials had high percentage of ricinoleic acid (79-90 %). The castor seed pulp showed high percentage of glutamic acid (15 %), arginine (11 %) and triptophane (9 %). The percentage of the other amino acids varied between 2 and 7 %. The results of the chemical characterization of the EPL and of the residues were mainly dependent on the pH during protein extraction. It was observed an increase of the protein concentration (64-68 %) and of the ash (13-19 %), within the EPL, due to the pH raise (10 - 12), independently of the type of alkaline agent. The residues of the EPL extraction showed protein, ash and fiber content varying between 20 and 34%, 12 and 17% e 39 and 42%, respectively. Concerning the mineral profile of the EPL, an increase of Na and of Mn was observed with the increase of pH for both alkaline agents. The mineral profile of the residues showed a significant increase of the Na and the K content with the increase of the pH of the extraction solution, adjusted with NaOH and KOH, respectively. The ricinoleic acid content was lower in the residue, and higher in the EPL, obtained at pH 12 using NaOH. The amino acid composition of the residues was affected by the pH only at pH 12. The higher content of amino acid in the EPL and in the residues was of glutamic acid, triptophane and arginine. The EPL and the residues of the protein extraction showed low allergenic capacity evaluated by the mastocyte degranulation test obtained from the peritoneal wash of rats. The electrophoresis profile of the EPL and of the residues showed higher content of proteins with molecular weight varying between 29-36 kDa, followed by proteins with molecular weight around 20 kDa and lower proportion of proteins with molecular weight between 45 and 66 kDa. Ricin was not observed in the residues obtain at pH 12. The results of all the experiments of protein extraction of the castor seed pulp allowed to select the best extraction parameters: temperature = 50 °C, stirring speed = 400rpm, castor seed pulp concentration in the extraction solution = 20 %, pH = 12 and alkaline agent = NaOH. In conclusion, through the alkaline protein extraction process it was possible to achieve a protein concentrate with interesting properties for biodegradable material production (agriculture and biofilms), and of ricinless residues which still have high content of proteins and fiber that can be used for animal feed.

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.

En studie i hur känsligheten avtar från ytan hos biosensorer med höga frekvenser presenteras. Med ny teknologi som avancerade elektroakustiska tunnfilms komponenter, så kallade FBARs, blir tidigare outforskade områden som decay längden möjliga att studera.

För att undersöka hur frekvenssvaret och känsligheten påverkas av interaktioner långt ut från en sensoryta används proteinkemi. Ett protokoll har optimerats innehållande aktivering med EDC/NHS och fibrinogen för att säkerställa en jämn tjocklek och fördelning av ett adsorberat proteinlager över en yta.

Dessa ytor kontrollerades först med hjälp av ellipsometri och sedan i ett QCM instrument. Alla experiment med de högfrekventa FBAR sensorerna utfördes vid Ångströmslaboratoriet i Uppsala där pågående forskning inom området finns.

Resultaten bekräftar teorin om en avtagande känslighet i och med ett ökat avstånd från ytan. En experimentell genomförd och beräknad tjocklek för decay längden uppskattades som inte helt stämde överens med den teoretiskt beräknade.

En ny term föreslås då frekvenssvaret hos en biosensor planar ut. Detta är en effekt som sker vid dubbla tjockleken av den teoretisk beräknade tjockleken av decay längden och har fått namnet; detection length. Efter denna längd eller gräns observeras en inverterad signal som det än så länge inte finns någon förklaring till.

A study of the sensitivity decrease of biosensors working at high frequencies is presented. With new technology such as film bulk acoustic resonators (FBAR), issues like the decay length is no longer irrelevant theory but may cause limitation in the system as well as it offers new detection possibilities.

To investigate the frequency response and sensitivity, layer-on-layer construction chemistry was used. A protocol involving activation with EDC/NHS and coupling chemistry with fibrinogen was optimized to ensure accurate thickness and uniformly distribution of each layer over the surface.

Surfaces were characterized using null ellipsometry and the protocol was tested in a traditional quartz crystal microbalance (QCM). Experiments with the FBAR were preformed at the Ångström laboratory in Uppsala were there is ongoing research and development in FBAR technology.

The results confirmed the theory of decreasing frequency and sensitivity further out from the surface. An experimental and estimated thickness was calculated which to some extent correlates to the theoretically calculated decay length.

A new terminology is suggested when the frequency levels off. It occurs approximately at twice the distance and thickness of the theoretically calculated decay length and is given the name; detection length. Beyond the detection length an inverted signal is observed which cannot yet be explained for.

No presente estudo objetivou-se avaliar o desempenho do etanol e isopropanol, absoluto e azeotrópico, na extração de óleo de macadâmia e milho. Nesse sentido, foram realizados experimentos de extração em batelada para determinar os efeitos do tipo de solvente e temperatura sobre o índice de retenção, rendimento de extração, qualidade do óleo e fração proteica. Assim, torta parcialmente desengordurada de macadâmia sem pré-tratamento prévio à etapa de extração (teor de lipídios 57,4 ± 0,7 % em base seca) foi submetida a extração, nas temperaturas de 60, 75 e 90 °C, enquanto as extrações de óleo do gérmen de milho em pellets (teor de lipídios 12,61 ± 0,05 % em base seca) foram realizadas a 50, 60, 70 e 80 °C. Os experimentos de extração em correntes cruzadas foram realizados a 70 e 75 °C para o gérmen de milho e a macadâmia, respectivamente. Os resultados mostraram que o rendimento de extração de óleo de macadâmia e milho é influenciado pelo tipo solvente, temperatura e pré-tratamento do material, sendo que com isopropanol absoluto, a 90 °C, foi possível extrair 66 % do óleo de macadâmia e a 80 °C, 87 % do óleo de milho. O índice de retenção de solução aderida as fibras (IR) foi impactado pelo tipo de solvente e características da matéria-prima, obtendo-se valores de IR maiores nas fases rafinado de macadâmia. A composição em ácidos graxos dos óleos de gérmen de milho extraídos com os solventes alcoólicos foi típica do óleo de milho, com predominância dos ácidos graxos oleico (34 %) e linoleico (48 %). As fases rafinado dos dois materiais apresentaram teor de proteínas maior que as matérias-primas iniciais, no entanto as condições do processo de extração impactaram negativamente no índice de solubilidade de nitrogênio, tornando inviável a produção de concentrado proteico de gérmen de milho. Para o óleo de milho a extração de tocoferóis e tocotrienóis foi maior com etanol, enquanto que com isopropanol conseguiu-se uma maior extração de carotenoides, o que pode ser decorrente da polaridade destes compostos bioativos. Os óleos de macadâmia extraídos com os solventes alcoólicos apresentaram maior estabilidade oxidativa (mais de 37 horas) quando comparados ao óleo bruto obtido por prensagem industrial (11,7 horas), sendo possível inferir que o uso dos solventes alcoólicos possibilita a extração de compostos que contribuem para o aumento da estabilidade do óleo. Nos experimentos em correntes cruzadas foi observado que para desengordurar a macadâmia com etanol e isopropanol absoluto são necessários mais de quatro estágios de contato, enquanto para o gérmen de milho dois estágios são suficientes. Em relação aos concentrados proteicos de macadâmia, o rendimento foi maior com os rafinados oriundos das extrações com isopropanol absoluto (13 %). Estes concentrados apresentaram boa capacidade de retenção de água e óleo (aproximadamente 2 g/g de concentrado) e estabilidade e atividade de emulsão na faixa de 56 a 59 %, desta forma pode-se sugerir que estes produtos apresentam potencial para seu aproveitamento na indústria de alimentos. A partir dos resultados pode-se inferir que a utilização dos solventes alcoólicos na extração de óleo de milho e macadâmia é tecnicamente viável, no entanto o desempenho dos solventes alcoólicos é influenciado pelas características da matéria-prima e pelo pré-tratamento do material sólido prévio à etapa de extração, obtendo-se maiores rendimentos de extração de óleo com o gérmen de milho em pellets. Também é possível inferir que os solventes em grau absoluto impactam menos na solubilidade das proteínas, permitindo a produção de concentrados proteicos de macadâmia com propriedades funcionais adequadas para seu uso na indústria de alimentos.
The objective of this study was to evaluate the performance of ethanol and isopropanol, absolute and azeotropic, in the extraction of macadamia and corn oil. Accordingly, extraction experiments were performed in batch to determine the effect of solvent type and temperature on retention index, extraction yield, quality indices of oil and protein fraction. Thus, partially defatted macadamia meal without pre-treatment prior to the extraction step (lipid content 57.4 ± 0.7 % on dry basis) was submitted to extraction at temperatures of 60, 75 and 90 °C, while oil extractions of corn germ in pellets (lipid content 12.61 ± 0.05 % on dry basis) were performed at 50, 60, 70 and 80 °C. The crosscurrent extraction experiments were performed at 70 and 75 °C for corn germ and macadamia, respectively. The results showed that the oil extraction yield of macadamia and corn is influenced by the solvent type, temperature and pre-treatment of the material, being that with absolute isopropanol at 90 °C, it was possible to extract 66 % of the macadamia oil and 87 % of the corn oil at 80 °C. The retention index of the solution adhered to the fibers (RI) was affected by the type of solvent and characteristics of the raw material, obtaining higher values of RI in the raffinate phases of macadamia. The fatty acid composition of corn germ oils extracted with alcoholic solvents was typical of corn oil, with predominance of oleic acid (34 %) and linoleic acid (48 %). The raffinate phases of the two materials showed higher protein content than the initial raw materials, however the conditions of the oil extraction process had a negative impact on the nitrogen solubility index, turning the production of protein concentrate from corn germ infeasible. For corn oil the extraction of tocopherols and tocotrienols was higher with ethanol, whereas with isopropanol a greater extraction of carotenoids was obtained, which can be due to the polarity of these bioactive compounds. Macadamia oils extracted with the alcoholic solvents showed higher oxidative stability (more than 37 hours) when compared to crude oil obtained by industrial cold pressing (11.7 hours). It is possible to infer that the use of alcoholic solvents enables to extract compounds that contribute to the increase of oil stability. In the crosscurrent extraction experiments it was observed that to produce defatted macadamia with absolute ethanol and isopropanol more than four stages of contact are required, whereas for the corn germ two stages are sufficient. Regarding protein concentrates of macadamia, the yield was higher with raffinate phases coming from the extractions with absolute isopropanol (13 %). These concentrates showed good water and oil retention capacity (approximately 2 g/g of concentrate) and emulsion stability and activity in the range of 56 to 59 %, therefore it can be suggested that these products have potential for their use in the food industry. From the results it can be inferred that the use of the alcoholic solvents in the extraction of corn and macadamia oil is technically feasible, however the performance of the alcoholic solvents is influenced by the characteristics of the raw material and by pre-treatment of the solid material prior to the extraction step, obtaining the highest oil extraction yields with the corn germ in pellets. Also, it is possible to infer that the solvents in absolute degree impact less on the solubility of the proteins, enabling the production of macadamia protein concentrates with functional properties suitable for use in the food industry.

Hydrogen bonds are directional, non-covalent interactions between hydrogen and electronegative atoms. Although generally weak, these interactions are critical to the stability of many biological systems including proteins and DNA. This dissertation explores small molecules in which an intramolecular hydrogen bond is the key determinant of conformation. Chapter 1 introduces the protein Grb2 SH3C, details its role in cancer signalling, and delineates the idea of peptidomimetics—small molecules which are functionalized to mimic the structure of a peptide and disrupt protein-protein interactions. Chapter 2 describes a virtual screen for binders to Grb2 SH3C. From a library of 6.3 million compounds, 34 were tested in vitro and two found to bind to the protein in two orthogonal assays. Chapter 3 describes mimics of the polyproline II helix using a benzoylurea scaffold. A small library of these compounds was synthesized and tested for binding to Grb2 SH3C using SPR, a competition assay, and NMR. Chapter 4 describes attempts to mimic a 310 helix using benzamide-based peptidomimetics. The synthesis and in vitro evaluation of these molecules as ligands of Grb2 SH3C is described. Chapter 5 uses quantum chemical calculations to assess the energies of a series of molecular switches. These calculations benchmark a range of modern density functional theory calculations, and attempt to quantify the accuracy of these methods for a large, flexible system. The role of solvation, entropy, geometry, and torsional angles are assessed in accurately calculating the energies of the critical hydrogen bonds.

The proteins wheat gluten and gelatin were tested for use in biocomposites and soft actuating materials, respectively. In Chapter II, the self-assembly mechanism of trypsin hydrolyzed wheat gluten (THWG) into rigid β-sheets was applied to an aqueous polyvinyl alcohol (PVA) environment. Aqueous PVA was used in order to determine the effects of an aqueous environment other than pure water on THWG self-assembly kinetics and to realize the potential use of THWG as a nanofiller in polymer matrices. THWG was able to self-assemble into anisotropic spikes and agglomerates of spikes called "pompons" through hydrophobic interactions. THWG self-assembly kinetics were retarded in aqueous PVA solutions compared to water, with the highest molecular weight PVA solution showing the slowest self-assembly kinetics. Chapters III and IV explore the potential of gelatin hydrogels for use in soft actuators. A gelatin bilayer system was designed where an active layer swelled more than a passive layer to cause the system to bend/actuate in response to an environmental stimulus. In Chapter III, gelatin layers were chemically crosslinked to different degrees with glutaraldehyde to achieve bilayer bending when placed in water. Curvature of the bilayer system was found to be dependent on the difference in volume swell ratio between the two layers. It was determined that maximum bending occurred when the passive layer swelled to 60% of the swelling of the active layer. Addition of pre-gelatinized starch to the active layer increased layer swelling and bilayer curvature. Treating the starch containing bilayer with -amylase returned the bilayer to its original shape. In Chapter IV, a pH responsive gelatin bilayer was constructed using Type A and Type B gelatin. Type A and Type B gelatin gels had different chemical properties and swelled to different volumes based on the gel solution pH. Bilayers constructed from Type A and Type B gelatin exhibited different degrees of bending when placed in various pH solutions with maximum curvature occuring at pH 10. A cyclic actuator could be formed when the bent bilayers were placed in a minimum of 0.01M NaCl solution. Placement in salt solution resulted in the unbending of the bilayer. Overall, this work demonstrated the various applications of proteins as functional and green materials.
Doctor of Philosophy
The majority of plastics consist of synthetic polymers derived from oil that cannot be broken down by the environment (i.e., not biodegradable). Research is underway to develop sustainable, biodegradable materials. Proteins are a biological polymer that have a wide range of chemical, structural, and functional properties; for this reason they are an excellent source material for use in the design of environmental friendly materials. In Chapter II, the ability of wheat gluten protein to self-assemble into rigid, nanosized structures is used to explore the potential of the protein to be used as a biodegradable nanofiller. A nanofiller is added to various materials in order to improve the overall mechanical properties of the material. Wheat gluten is self-assembled in an aqueous polymer environment. The results show that the polymer environment stunts or slows down the self-assembly rate of the protein compared to a pure water environment. Nanometer sized spikes form in the polymer solutions, indicating wheat gluten could be used as a nanofiller in certain materials. Chapters III and IV explore the use of gelatin proteins for applications in soft robotics. Soft robots and their moveable parts, called soft actuators, are deformable and respond to changes in the environment such as pH, light, temperature, etc. For this reason, soft robots are considerable adaptable compared to traditional rigid robots. Designing a soft actuator from gelatin gels would result in a “smart” material that is biocompatible and biodegradable. A gelatin soft actuator is created using a bilayer design in which one layer of the bilayer swells more than the other layer causing the entire system to bend/actuate. Depending on how the bilayer system was fabricated, bending could be achieved based on stimuli such as the presence of water, the presence of a substrate and enzyme, and changes in pH. Overall, this dissertation demonstrates the extraordinary potential for the use of proteins in designing sustainable materials.

The inherent design freedom of protein engineering and recombinant protein production enables specific tailoring of protein structure, function, and properties. Two areas of research where protein engineering has allowed for many advances in biomedical materials include the design of novel protein scaffolds for molecular recognition, as well as the use of recombinant proteins for production of next generation biomaterials. The main focus of my dissertation was to develop new biomedical materials using protein engineering. Chapters three and four discuss the engineering of repeat proteins as bio-recognition modules for biomedical sensing and imaging. Chapter three provides an overview of the most recent advances in engineering of repeat proteins in the aforementioned field. Chapter four discusses my contribution to this field. We have designed a de novo repeat protein scaffold based on the consensus sequence of the leucine rich repeat (LRR) domain of the NOD family of cytoplasmic innate immune system receptors. Innate immunity receptors have been described as pattern recognition receptors in that they recognize "global features" of a family of pathogens versus one specific antigen. In mammals, two main protein families of such receptors are: extracellular Toll-like receptors (TLRs) and cytoplasmic Nucletide-binding domain- and Leucine-rich Repeat-containing proteins (NLRs). NLRs are defined by their tripartite domain architecture that contains a C-terminal LRR (Leucine Rich Repeat) domain, the nucleotide-binding oligomerization (NACHT) domain, and the N-terminal effector domain. It is proposed that pathogen sensing in NLRs occurs through ligand binding by the LRR domain. Thus, we hypothesized that LRRs would be suitable for the design of alternative binding scaffolds for use in molecular recognition. The NOD protein family plays a very important role in innate immunity, and consequently serves as a promising scaffold for design of novel recognition motifs. However, engineering of de novo proteins based on the NOD family LRR domain has proven challenging due to problems arising from protein solubility and stability. Consensus sequence design is a protein design tool used to create novel proteins that capture sequence-structure relationships and interactions present in nature in order to create a stable protein scaffold. We implement a consensus sequence design approach to develop proteins based on the LRR domain of NLRs. Using a multiple sequence alignment we analyzed all individual LRRs found in mammalian NLRs. This design resulted in a consensus sequence protein containing two internal repeats and separate N- and C- capping repeats named CLRR2. Using biophysical characterization methods of size exclusion chromatography, circular dichroism, and fluorescence, CLRR2 was found to be a stable, monomeric, and cysteine free scaffold. Additionally, CLRR2, without any affinity maturation, displayed micromolar binding affinity for muramyl dipeptide (MDP), a bacterial cell wall fragment. To our knowledge, this is the first report of direct interaction of a NOD LRR with a physiologically relevant ligand. Furthermore, CLRR2 demonstrated selective recognition to the biologically active stereoisomer of MDP. Results of this study indicate that LRRs are indeed a useful scaffold for development of specific and selective proteins for molecular recognition, creating much potential for future engineering of alternative protein scaffolds for biomedical applications. My second research interest focused on the development of proteins for novel biomaterials. In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues, as well as participates in important regulatory functions such as cell migration and proliferation and protein signalling. The aforementioned properties along with keratins' inherent capacity for self-assembly poise it as a promising scaffold for regenerative medicine and tissue engineering applications. However, due to the extraction process used to obtain natural keratin proteins from natural sources, protein damage and formation of by-products that alter network self-assembly and bioactivity often occur as a result of the extensive processing conditions required. Furthermore, natural keratins require exogenous chemistry in order to modify their properties, which greatly limits sequence tunability. Recombinant keratin proteins have the potential to overcome the limitations associated with the use of natural keratins while also maintaining their desired structural and chemical characteristics. Thus, we have used recombinant DNA technology for the production of human hair keratins, keratin 31 (K31) and keratin 81 (K81). The production of recombinant human hair keratins resulted in isolated proteins of the correct sequence and molecular weight determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectrometry. Proteins with no unwanted sequence truncations, deletions, or mutations indicate recombinant DNA technology can be used to reliably generate full length keratin proteins. This allows for consistent starting materials with no observable impurities or undesired by-products, which combats a major challenge associated with natural keratins. Additionally, recombinant keratins must maintain the intrinsic propensity for self-assembly found in natural keratins. To test the propensity for self-assembly, we implemented size exclusion chromatography (SEC), dynamic light scattering (DLS), and transmission electron microscopy (TEM) to characterize K31, K81, and an equimolar mixture of K31 and K81. The results of the recombinant protein characterization reveal novel homo-polymerization of K31 and K81, not previously reported, and formation of characteristic keratin fibers for the K31 and K81 mixture. Therefore, recombinant K31 and K81 retain the intrinsic biological activity (i.e. self-assembly) of natural keratin proteins. We have also conducted a comparative study of recombinant and extracted heteropolymer K31/K81. Through solution characterization and TEM analysis it was found that use of the recombinant heteropolymer allows for increased purity of starting material while also maintaining self-assembly properties necessary for functional use in biomaterials design. However, under the processing condition implemented, extracted keratins demonstrated increased efficiency of assembly. Through each study we conclude that recombinant keratin proteins provide a promising solution to overcome the challenges associated with natural protein materials and present an exceptional design platform for generation of new biomaterials for regenerative medicine and tissue engineering.
Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2012.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student submitted PDF version of thesis.
Includes bibliographical references (p. 109-124).
Protein-protein interactions (PPIs) play a central role in all biological processes. Akin to the complete sequencing of genomes, complete descriptions of interactomes is a fundamental step towards a deeper understanding of biological processes, and has a vast potential to impact systems biology, genomics, molecular biology and therapeutics. PPIs are critical in maintenance of cellular integrity, metabolism, transcription/ translation, and cell-cell communication. This thesis develops new methods that significantly advance our efforts at structure- based approaches to predict PPIs and boost confidence in emerging high-throughput (HTP) data. The aims of this thesis are, 1) to utilize physicochemical properties of protein interfaces to better predict the putative interacting regions and increase coverage of PPI prediction, 2) increase confidence in HTP datasets by identifying likely experimental errors, and 3) provide residue-level information that gives us insights into structure-function relationships in PPIs. Taken together, these methods will vastly expand our understanding of macromolecular networks. In this thesis, I introduce two computational approaches for structure-based proteinprotein interaction prediction: iWRAP and Coev2Net. iWRAP is an interface threading approach that utilizes biophysical properties specific to protein interfaces to improve PPI prediction. Unlike previous structure-based approaches that use single structures to make predictions, iWRAP first builds profiles that characterize the hydrophobic, electrostatic and structural properties specific to protein interfaces from multiple interface alignments. Compatibility with these profiles is used to predict the putative interface region between the two proteins. In addition to improved interface prediction, iWRAP provides better accuracy and close to 50% increase in coverage on genome-scale PPI prediction tasks. As an application, we effectively combine iWRAP with genomic data to identify novel cancer related genes involved in chromatin remodeling, nucleosome organization and ribonuclear complex assembly - processes known to be critical in cancer. Coev2Net addresses some of the limitations of iWRAP, and provides techniques to increase coverage and accuracy even further. Unlike earlier sequence and structure profiles, Coev2Net explicitly models long-distance correlations at protein interfaces. By formulating interface co-evolution as a high-dimensional sampling problem, we enrich sequence/structure profiles with artificial interacting homologus sequences for families which do not have known multiple interacting homologs. We build a spanning-tree based graphical model induced by the simulated sequences as our interface profile. Cross-validation results indicate that this approach is as good as previous methods at PPI prediction. We show that Coev2Net's predictions correlate with experimental observations and experimentally validate some of the high-confidence predictions. Furthermore, we demonstrate how analysis of the predicted interfaces together with human genomic variation data can help us understand the role of these mutations in disease and normal cells.
by Raghavendra Hosur.
Ph.D.

La technique de nanoprécipitation est une méthode simple et reproductible pour la synthèse de nanocapsules à coeur huileux recouvertes d’une enveloppe de polymères hydrophiles réticulés (polysaccharides, glycopolymères vinyliques…) en une seule étape. Grâce à leur biocompatibilité, leur biodégradabilité et leur activité biologique adaptables, les protéines constituent une autre grande famille de biopolymères d’intérêt pour des applications dans le domaine de l’encapsulation. Cependant, la production de nanocapsules protéiques par nanoprécipitation n’a jamais été décrite. Dans ce contexte, l’objectif principal de ce travail de thèse a été l’évaluation du potentiel d’une famille de protéines, les Suckerines, pour le procédé de nanoprécipitation. Les Suckerines sont une famille de protéines issues des dents décorant les ventouses du calamar géant Humboldt avec de prometteuses applications dans le domaine biomédical. Ces protéines possèdent une structure modulaire de type copolymère à bloc capable de former des feuillets bêta conférant de bonnes propriétés mécaniques. Les suckerines étant solubles dans une solution tampon composée d’acide acétique (pH 3) mais fortement agrégées dans les conditions de pH (valeurs comprises entre 5 et 10) classiquement utilisées pour la préparation de nanocapsules à coeur huileux par basculement de solvant, nous avons finalement choisi d’explorer la nanoprécipitation des protéines par salt shifting et donc la préparation de nanoparticules protéiques. L’utilisation du persulfate d’ammonium comme agent de coacervation et précurseur de radicaux et du tris(2,2′ bipyridyl)dichlororuthenium(II) hexahydrate a permis de produire des nanoparticules de suckerine de tailles modulables (100-185 nm de diamètre). Ces nanoparticules présentent des structures secondaires type feuillets bêta qui sont à l’origine du module de Young très élevé observé pour ces nano-objets (de l’ordre de grandeur du GPa). Une protéine de fusion, soluble en milieu aqueux à pH 7 a spécialement été conçue par voie de recombinaison dans le but de générer des nanocapsules protéiques par nanoprécipitation. Cette protéine (suckerine-soie) est formée d’un bloc central de peptide dérivé de suckerine de calamar promouvant une stabilité structurelle et deux blocs terminaux issus de fibroïnes de soie qui permettent à la protéine de fusion d’être soluble à un pH physiologique. Ce design moléculaire a permis la fabrication de nanocapsules remplies respectivement de hexadécane ou de miglyol avec une enveloppe de suckerine-soie et de tailles de l’ordre de grandeur de 190 à 250 nm. Finalement, aspirant à encapsuler un principe actif anti-cancéreux dans les nanocapsules à base de glycogène, nous avons développé un protocole où la méthode de nanoprécipitation est utilisée pour produire des nanoparticules de prodrogue entourés de glycogène
The nanoprecipitation technique is a reliable route to synthesize oil filled nanocapsules with shells made of hydrophilic polymers such as polysaccharides and vinyl based glycopolymers in a one pot procedure. Thanks to their biocompatibility, biodegradability and tunable biological activity, proteins are another promising class of materials for encapsulation purposes. However, the generation of proteinaceous nanocapsules by nanoprecipitation has never been reported. In this context, the main objective of this PhD was to evaluate the potential of a family of proteins, the Suckerins, in nanoprecipitation processes. Suckerins are a family of proteins found in the sucker ring teeth of the giant Humboltd squid with promising biomedical applications. These proteins possess a modular, block copolymer like structure capable of forming β-sheets responsible for good mechanical properties. The suckerin proteins are not soluble at a pH range between 5 and 10, a requirement of the nanoprecipitation technique. However, they can be solubilized using aqueous buffers at pH 3 containing acetic acid. Other ways of precipitating the protein were explored in this manuscript with salt shifting using ammonium persulphate as coacervation agents being capable of generating 100 nm nanoparticles. These nanoparticles presented the β sheet secondary structure which resulted in Young modulus in the GPa range. A fusion protein that could be solubilized in aqueous solutions at pH 7, and therefore be used in the nanoprecipitration process, was recombinantly produced. The protein (suckerin silk) is formed by a central squid suckerin-derived peptide block that provides structural stability and both termini from silk fibroins that make the modular protein highly soluble at physiological pH. This molecular design allowed the fabrication of hexadecane and miglyol filled nanocapsules with suckerin silk shells and sizes in the range 190 – 250 nm. Finally, aiming to encapsulate an anti cancer drug in glycogen nanocapsules we developed a protocol where the nanoprecipitation process is used to generate glycogen coated prodrug nanoparticles

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references (pages [121]-140).
Gold nanoparticles with amphiphilic surface functionalization have been shown to spontaneously fuse with lipid bilayers through a non-endocytic mechanism that generates minimal membrane perturbation. The membrane translocation capability of these nanoparticles makes them attractive candidates for engineering clinical applications that operate on a single-cell resolution. In particular, the physiochemical similarity between these nanoparticles and membrane-bound and free-circulating proteins suggests a possibility for designing nanostructures that can function as synthetic alternatives to proteins. In this thesis, we demonstrate how molecular simulation techniques have allowed us to tackle this engineering challenge and develop nanoparticles that can modulate fusion between lipid membranes, transport hydrophobic small molecules to lipid-bound compartments, and modify the permeability of lipid membranes. These are concrete realizations of nanoparticles functioning as protein mimics, and unlock new avenues of research on how nanomaterials can be designed from first principles to perform targeted functions in biological systems.
by Mukarram Ahmad Tahir.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Materials Science and Engineering

Inspired by the core-shell composite structures found in nature, a range of protein based composites have been developed. These materials were made using synthetic approaches, which utilized the native protein architecture as an initiation point and size constrained reaction vessel for the piecewise formation of the second material. In the first illustration of this approach, a protein-P t composite was formed, where the protein cage has been modified to include a metal binding moiety for improved synthesis of metallic P t nanoclusters, which were shown to be an active H ₂ catalyst. This composite was analyzed by native mass spectrometry to determine the number of P t ions bound prior to mineralization and to measure the distribution of species after mineralization, which provided a unique view into the mineralization process. The second illustration was a material synthesized using the cage-like protein architecture as an internal guiding synthetic scaffold for the formation of a coordination polymer core inside the protein cage. The construction of this coordination polymer was unusual in that unlike normal coordination polymer synthesis, coordination of the metal preceded formation the ditopic ligands, which were afterwards completed using azide-alkyne click chemistry. Finally, a collection of protein-polymer composites were developed, which utilized a living radical polymerization method, atom transfer radical polymerization, to form internal polymer cores. By labeling one of these protein-polymer constructs with a Gd based MRI contrast agent a material with vastly improved relaxivity was made. The development of each of these three types of composites served to improve our understanding of the natural systems, from which they are derived, and provide a basis for further development of advanced multicomponent nanomaterials. 'Co-authored by Md Joynal Abedin, Masaki Uchida, Lars Liepold, Craig C. Jolley, Mark Young, Trevor Douglas, Shefah Qazi, Gregory J. Bedwell, Ben LaFrance, and Peter E. Prevelige, Jr.'

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Materials Science and Engineering, 2015.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 165-177).
Amyloid and amyloid-like proteins are a broad class of misfolded protein structures known for their their roles in a variety of neurodegenerative diseases, but also for their impressive mechanical properties and their propensity to self-assemble at diverse length scales. These properties make amyloid and amyloid-like proteins excellent candidate materials for the design of engineered functional biomaterials. However, many of the fundamental structure-property relationships which could guide the design of amyloid-based functional materials for various applications are not well understood. In this thesis, a multiscale modeling and simulation approach is used to investigate these structure-property relationships at multiple length scales. Full atomistic simulations are used to study the tensile and bending response of single fibrils, as well as the inter-fibril interaction strength. It is found that in tension, the specific geometry of the fibrils does not significantly influence the deformation behavior, but the mechanical properties, most notably the tensile strength, depends strongly on the areal density of hydrogen bonds in the fibril cross-section. The mechanical response at the molecular scale is used to guide the development of a coarse-grained description of amyloid and amyloid-like fibrils. Next, the adhesive behavior of amorphous polymers is studied to identify design principles which enhance adhesive performance and could be applied to aid in the design of amyloid-based adhesives, an exciting potential functional role for amyloid-based biomaterials. Finally, mesoscale structures are investigated including a nanowire-like geometry and adhesive films. These studies demonstrate that the mechanics of larger scale amyloid based structures are largely determined by the inter-fibril interactions; the specific intra-fibril properties become less significant at larger scales. The results presented in this thesis form the foundation for the development of basic materials selection criteria to aid in the design of functional amyloid-based biomaterials for diverse applications.
by Max Isaac Solar.
Ph. D.

Proteomics plays an important role in the recognition of diseases and the understanding of biological processes. Sample preparation is a bottleneck in systems for chemical analysis and it is a required step in proteomics in order to remove interferences and preconcentrate the proteins. In addition, protein reduction and alkylation before digestion is a required step in proteomics to facilitate protein unfolding and increase the efficiency of enzymes in digesting proteins. The purpose of this study was to develop new techniques to address some of the shortcomings of current sample preparation methods, and provide short sample preparation time. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transfer compared with common packed beds. This study has focused on the use of organic polymer- and inorganic silica-based monolithic materials for protein sample preparation. The organic polymer monolith used in this study was a butyl methacrylate-co-ethylene dimethacrylate (BuMA-co-EDMA) monolith that was fabricated inside the borosilicate tube using photoinitiated polymerisation. The porous properties of the fabricated monolith were controlled by adjusting the composition of the porogenic solvent in the polymerisation mixture. The results indicated that using MeOH/1-propanol as a porogenic solvent produced a polymer-based monolith with high surface area (56.89 m² g¯¹); however, it lacked the desired permeability and porosity when fabricated inside a glass microchip. Evaluation of its performance was carried out by extraction of four standard proteins that were insulin, cytochrome C, myoglobin, and hemoglobin and the extraction recovery was in the range (79.1-98.4 %). A monolithic silica rod was fabricated without cracks inside a heat shrinkable tube and then compared with the same material whose surface has been modified with octadecyl groups in order to use them for preconcentration/extraction of proteins. Their performance was evaluated using eight standard proteins, namely insulin, cytochrome C, lysozyme, myoglobin, β-lactoglobulin, ovalbumin, hemoglobin, and bovine serum albumin. The results show that recovery of the proteins was achieved by both columns with variable yields; however, the octadecylated silica monolith gave higher recoveries (92.7 - 109.7%) than the non-modified silica monolith (25.5 - 97.9%). This was followed by a new process for the fabrication of a silica-based monolith inside a glass microchip, which was successfully developed for use in microchip-based solid phase extraction of proteins. This was achieved by placement of the monolithic silica disk inside the extraction chamber in the base plate of the microchip, followed by thermal bonding of the two plates of the glass microchip at 575 °C for 3 hours. By doing this, the problem of shrinkage in the silica skeleton during preparation was avoided completely. The monolithic silica disk inside the glass microchip was subsequently modified with octadecyl groups for increased protein binding capacity. The performance of the microchip was evaluated using the extraction of standard proteins mixed with a high concentration of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The results show that the octadecylated silica monolith was permeable, has the ability to remove impurities, and achieved a high extraction recovery of the proteins (94.8-99.7%) compared with conventional octadecylated silica particles (48.3-91.3%). The intra-batch and inter-batch RSDs were in the range of 2.0-4.5% and 2.9-6.4%, respectively. Comparison between the fabricated device and a commercial cartridge for the preconcentration of proteins in skimmed cows milk and hen egg white showed the ability of the device to successfully enrich protein mixtures from more realistic samples. This new microfluidic device for protein extraction may find an application in the area of proteomic research. A novel approach for immobilisation of the reducing reagent on the surface of the silica-based monolith in order to use it for protein reduction and alkylation was successfully developed. This was carried out by silanisation of the surface of the silica-based monolith with (3-aminopropyl)triethoxysilane (APTES), followed by immobilisation of the reducing reagent, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) on the surface of the amino-bonded silica monolith. The fabricated monolith was characterised using IR spectroscopy, EDX analysis, BET model, and measuring the contact angle of deionised water. The fabricated monolith was evaluated for its use in protein reduction and alkylation in one single step at 60 °C by injection of a mixture consisting of 40 μL denatured protein and 60 μL iodoacetamide solution into the fabricated microchip, followed by using MALDI-TOF-MS instrument for qualitative confirmation. The results show that the fabricated microchip-based silica monolith has the ability to reduce and alkylate insulin in 30 min, and lysozyme in 45 min. Although this method was shown to require sample desalting to remove denaturant (urea) and the performance of the fabricated monolith had low intra-chip reproducibility, the method was simple, reduced the risk of contamination, decreased the number of processing steps, and results in lower amounts of the sample and reagents compared with the conventional techniques for proteomics sample preparation. More work is required to fully optimise this approach to protein sample preparation.

Structures in nature exhibit unique and complex architectures whose order propagates from nano- (10-9 m) to macro-scales (mm to m). These structures give rise to a rich diversity of adaptive function that allows for life in all environments on Earth. This complex functionality has driven research into bio-inspired materials where scientists investigate the complex relationship between sequence, structure and function of these materials. A good illustrative example of the effect that hierarchical structure can have is a brick wall. Bricks are laid so that the layer on top is shifted in either direction by half of a brick. This alternating pattern is what gives the wall its strength. If a crack occurs in the mortar, it will only propagate until it hits a boundary (a neighboring brick). Designing nanostructures can have similar effects on materials we use every day. Some of the most prevalent are adhesives that mimic the structures on gecko feet, which allow them to stick to any surface. This work presents bottom-up design strategies for self-assembling protein materials whose hierarchical structure may prove useful in a variety of applications in soft-robotics and energy storage. Proteins are a useful class of molecules, because they contain a level of structural complexity beyond that of synthetic materials. They are an inherently 'green' material feedstock; made in a lab using microbes like E. coli. Additionally, with the ease and availability of genetic engineering techniques we can easily modify the structure. This is especially true for the class of proteins, repeat proteins, which are the focus of this manuscript. Repeat proteins comprise small repeated sequences which are structurally independent from each other and can be strung together to create open, extended architectures. Here we explore the self-assembly emergent properties of the consensus tetratricopeptide repeat (CTPR18) . We show that this protein assembles into highly ordered 1D and 2D arrays that are shape tunable based the molecular environment (solvents, charge, etc). These nanomaterials may prove useful as molecular recognition scaffolds. We further explore the hierarchical self-assembled films of CTPR18. These films form highly oriented lamellar structures that seemingly propagate the entire length of the films. These lamellae directly affect the materials mechanical properties. Accordingly, by changing the film casting conditions, we can impart a structural gradient in the film, which proves useful in tuning the water-induced bending motion of these films. Herein, we show the ability to change the speed and directionality of actuation by simply changing the underlying film morphology. Lastly, we show that these films are electroresponsive as well, owing this function to ion transport through the inherently charged character of CTPR18. These dual responsive materials may prove useful in soft robotics. Additionally we are beginning investigations into the usefulness of CTPR18 films as alternate materials for ion-transport materials like those used in lithium polymer (more commonly LiPo) and sodium-ion batteries.
PHD

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

Obesity is a major global and national problem with a variety of consequences and effects. The relationship of fetal programming with obesity has been discovered through both human and animal studies and it has been found that nutritional programming can predispose the developing offspring to obesity later in life. However, many aspects of this fetal programming have yet to be fully determined, for instance the environmental and metabolic factors involved. This thesis has investigated the programming effects of maternal low protein restriction (MLP) and maternal age (2 month, 4 month and 6-9 month old mothers) in the rat, looking at both immature (4 weeks old) and mature (9 months old) adult offspring. Postnatal high fat diets were fed for 9 weeks to exacerbate potential obesogenic effects. During the trials various measures were studied, body weight, feed intake, energy expenditure, metabolism, body composition, body fat distribution, activity and behaviour.

The importance of tissue protein reserves to lactating females attempting to sustain milk output under conditions of severe dietary protein restriction was investigated using rats. Four experiments were carried out to study the effect of variation in repletion of tissue protein reserves on lactational performance, rates of body protein mobilisation and changes in tissue protein metabolism involved in promoting protein mobilisation. The extent to which body protein reserves were capable of maintaining milk quantity and quality under such conditions was also considered. The lactation performance of multiparous, female Sprague-Dawley rats, offered isoenergetic diets (21 MJ GE/kg DM), was assessed from growth of a standardised litter of 12 pups. Variation in repletion of protein reserves at parturition was achieved by applying a period of protein restriction during the latter half of gestation. Changes in body composition were estimated from carcass analysis and rates of protein mobilisation were derived from serial slaughter experiments. Tissue protein synthesis was estimated in vivo using a flooding dose of [³H] phenylalanine and tissue Na+ , K+ -ATPase activity was measured polarographically in vitro. Milk samples were obtained following injection of oxytocin. Females offered a high protein diet (215 gCP/kg DM) during lactation exhibited an increase in both feed intake and lactational performance while not utilising their body protein stores. However, in rats offered imbalanced feeds (low protein/high energy) such an increase in intake was not apparent and dams were forced to draw upon their endogenous protein reserves in an attempt to sustain milk production. Between 15 and 22% of body protein was lost by dams assumed to be 'Fully' protein replete at parturition. When dietary protein was limiting, reductions in the size of the protein reserve had a significant inpact on a female's ability to sustain milk production, and dams which were initially 'Fully' replete supported greater (P< 0.05) litter growth during early lactation, due to a greater endogenous protein supply and feed intake (P< 0.05), than their 'Depleted' contemporaries.

Nanoparticles are increasingly important in biotechnology as they are extensively used as drug delivery carriers and in biosensors. In both these two contexts, protein-nanoparticle interactions are often involved. Proteins that are present in body fluids inevitably interact with nanoparticle based drug carriers and typically surround them forming the so called “protein corona”. Biosensors that are based on nanoparticles often have proteins deliberately attached to their surface, for example antibodies that bind specific analytes. The understanding of the assembly mechanisms at the protein-nanoparticle interface and the ability to engineer proteins that interact with nanoparticles in the desired way, are therefore two essential requisites for the future development of nano-medicines and nano-biosensors. In this work, we focused on the interaction of proteins with gold nanoparticles (GNPs). GNPs are available with a broad range of surface chemistries, suitable for the conjugation of many biomolecules. Although there are at least three decades of studies on gold colloids with different surface chemistries, there is still quite little known about what are the exact features of a protein that determine its adsorption onto gold. We developed methods to study this and applied them to characterise the adsorption on GNPs of Glutathione-S-Transerase (GST), which was reported previously as a protein that strongly binds gold. We determined its affinity and kinetics of binding and unravelled the mechanism of its thiol-mediated chemisorption. We found that GST binds to GNPs even more efficiently than other known gold-binding proteins, such as Bovine Serum Albumin (BSA). We concluded that GST could be considered a very useful gold-protein interface, especially considering that GST fusion is routinely used for affinity purification of recombinant proteins and therefore well established. We also fused self-assembling proteins to GST or chemically cross-linked them to BSA. The scope was to explore the feasibility of hierarchical and ordered assembly of designer proteins onto GNPs, with the ultimate goal of providing a convenient tool for modular assembly of proteins onto nanomaterials. It is known that proteins tend to denature and lose their function when in contact with GNPs, which is not optimal for biosensors or in nanomedicine. We found that it is possible to use GST or BSA to form a sacrificial layer on gold, which exposes linked, self-assembling proteins that are able to bind their counterpart, unaffected by the GNP surface. We reported two proof-of-concepts: the first based on mimics of the self-assembling neuronal SNARE proteins and the second based on the pair SpyCatcher/SpyTag, derived from Streptococcus pyogenes proteins and used in bio-conjugation for their ability to self-catalyse the formation of isopeptidic bonds. We believe that the novel methods and original results presented in this thesis apply to both the understanding and the engineering of the protein-nanoparticle interface and will be beneficial for the broad nanobiotechnology community. In fact, our findings have potential applications in a broad range of fields, spanning from the improvement of the circulation life-time of nanomedicines by preventing the binding of serum protein and opsonisation, to the improvement of the manufacturing of GNPs-based immune-biosensors such as those used in lateral flow devices.

The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.
Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 199-217).
Protein materials such as spider silk can be exceptionally strong, and they can stretch tremendously before failure. Notably, silks are made entirely of proteins, which owe their structure and stability to weak molecular interactions, in particular, hydrogen bonds (H-bonds). Beta-structures, a class of protein folds that employ dense arrays of H-bonds, are universal in strong protein materials such as silks, amyloids, muscle fibers and virulence factors. The biological recipe for creating strong, tough materials from weak bonds, however, has so far remained a secret. In this dissertation, size, geometry and deformation rate dependent properties of beta-structures are investigated, in order to provide a link between the nanostructure and mechanics of protein materials at multiple length scales. Large-scale molecular dynamics (MD) simulations show that beta-structures reinforce protein materials such as silk by forming H-bonded crystalline regions that cross-link polypeptide chains. A key finding is that superior strength and toughness can only be achieved if the size of the beta-sheet crystals is reduced to a few nanometers. Upon confinement into orderly nanocrystals, H-bond arrays achieve a strong character through cooperation under uniform shear deformation. Moreover, the size-dependent emergence of a molecular stick-slip failure mechanism enhances toughness of the material. Based on replica-exchange MD simulations, the first representative atomistic model for spider silk is proposed. The computational, bottom-up approach predicts a multi-phase material with beta-sheet nanocrystals dispersed within semi-amorphous domains, where the large-deformation and failure of silk is governed by the beta-structures. These findings explain a wide range of observations from single molecule experiments on proteins, as well as characterization studies on silks. Results illustrate how nano-scale confinement of weak bond clusters may lead to strong, tough polymer materials that self-assemble from common, simple building blocks.
by Sinan Keten.
Ph.D.