Le marquage protéique par fluorescence est une méthode de choix permettant d’étudier l’évolution des protéines depuis leur synthèse cellulaire jusqu’à leur dégradation, en plus de rendre possible leur localisation ainsi que la visualisation des interactions entre protéines. De cet intérêt certain ont découlé différentes techniques de marquage, dont celle présentement développée dans le groupe Keillor. Le principe de celle-ci repose sur la réaction entre deux maléimides portés par un fluorogène et une séquence peptidique cible, laquelle contient deux résidus cystéines séparés par une distance appropriée. Suite à cette double addition de thiols du peptide sur les maléimides du fluorogène, la fluorescence latente de ce dernier est régénérée, menant au marquage covalent de la protéine d’intérêt. Afin d’optimiser la spécificité et la sensibilité de cette méthode de marquage, la synthèse de nouveaux fluorogènes et l’étude de l’efficacité de quench de la fluorescence par les maléimides est présentement en cours dans les laboratoires du groupe Keillor. / The fluorescent labelling of proteins is a powerful approach for following the dynamic process of their cellular synthesis and degradation, in addition to determining their localization and protein-protein interactions. We have developed a ‘small molecule’-based labelling technique that is complementary to existing methods. Our fluorogenic approach relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest. Synthesis of new fluorogens and quench efficiency studies are presently taking place in the Keillor group laboratory in order to optimize the specificity and sensitivity of the labelling method.

Le marquage protéique par fluorescence est une méthode de choix permettant d’étudier l’évolution des protéines depuis leur synthèse cellulaire jusqu’à leur dégradation, en plus de rendre possible leur localisation ainsi que la visualisation des interactions entre protéines. De cet intérêt certain ont découlé différentes techniques de marquage, dont celle présentement développée dans le groupe Keillor. Le principe de celle-ci repose sur la réaction entre deux maléimides portés par un fluorogène et une séquence peptidique cible, laquelle contient deux résidus cystéines séparés par une distance appropriée. Suite à cette double addition de thiols du peptide sur les maléimides du fluorogène, la fluorescence latente de ce dernier est régénérée, menant au marquage covalent de la protéine d’intérêt. Afin d’optimiser la spécificité et la sensibilité de cette méthode de marquage, la synthèse de nouveaux fluorogènes et l’étude de l’efficacité de quench de la fluorescence par les maléimides est présentement en cours dans les laboratoires du groupe Keillor. / The fluorescent labelling of proteins is a powerful approach for following the dynamic process of their cellular synthesis and degradation, in addition to determining their localization and protein-protein interactions. We have developed a ‘small molecule’-based labelling technique that is complementary to existing methods. Our fluorogenic approach relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest. Synthesis of new fluorogens and quench efficiency studies are presently taking place in the Keillor group laboratory in order to optimize the specificity and sensitivity of the labelling method.

Les matériaux agroressourcés à base de gluten de blé présentent des propriétés mécaniques qui ne leurs permettent pas de concurrencer celles des plastiques usuels issus de la pétrochimie. Les objectifs de ce travail de thèse visent (i) à améliorer les propriétés d'élongation et de résistance des matériaux gluten pour atteindre celles des polymères courants et (ii) à maîtriser la réactivité du gluten au cours de l'élaboration des matériaux afin de pouvoir utiliser les procédés connus de la plasturgie. L'enjeu scientifique est de comprendre la réactivité du gluten sous l'effet des traitements thermomécaniques et les mécanismes régissant les propriétés mécaniques des matériaux. Les fonctions réactives visées sont les thiols/disulfures qui assurent la réticulation des protéines du gluten. Nous avons testé l'effet de bloqueurs de thiols de type maléimide mono- et bifonctionnels, de nature, de taille et d'hydrophobicité variées. Ces derniers ont éventuellement été bloqués par réaction de Diels-Alder. L'ajout d'additifs de type bismaléimide bloqué par réaction de Diels-Alder permet de différer la réticulation à l'étape de thermoformage et de substituer aux liens covalents habituels des liens thioéthers. L'ajout de cet additif permet de doubler l'élongation à la rupture du matériau gluten mais entraîne la chute de la rigidité. L'effet de l'ajout de molécules bis- et tétrathiols a également été testé. Ces additifs ont permis d'augmenter par plus de 1,5 fois l'élasticité des matériaux gluten. Une analyse multi-échelle (moléculaire par FTIR, macromoléculaire par SEC et macroscopique par test de traction ; le tout complété par une analyse DMTA) de la structure et des propriétés a montré que l'absence de gain en élasticité était due au maintien d'une organisation structurale majoritaire en hélices-α, qui est le propre du gluten natif. La création d'interactions interprotéiques par feuillets-β a été identifiée comme seule responsable du gain d'élasticité des matériaux, la formation d'agrégats protéiques par le biais de liaisons disulfures ou thioéthers ne jouant qu'un rôle secondaire. Un mécanisme réactionnel mettant en avant les conditions qui assurent la participation de toutes les classes de protéines du gluten à la constitution du réseau protéique est discuté. Deux nouvelles voies prometteuses de mélange avec du caoutchouc et copolymérisation par « grafting from » ont été explorées et restent à approfondir. / Wheat gluten can be used to make biomaterials. Nevertheless their mechanical properties are not competitive with commonly used petroleum-based plastics. The purposes of this work aim at (i) improving strain and strength properties of gluten-based materials in order to reach those of common polymers and (ii) controlling gluten reactivity during material process in order to use already well-known processes for manufacturing plastics. The scientific stakes are to understand gluten reactivity during thermo-mechanical treatments and the mechanisms which govern mechanical properties of materials. The reactive functions of gluten are thiols/disulfides which are responsible of gluten proteins crosslinking. The effect of thiol blocker molecules such as mono and bismaleimide of various nature, sizes and hydrophobicity was tested. These molecules were eventually blocked by Diels-Alder reactions. The addition of bismaleimide blocked by Diels-Alder reaction enables to postpone the crosslinking to the thermo-molding step and also to substitute disulfides bonds for thioether bonds. The addition of this additive succeeds in doubling the strain at break of gluten-based material but leads to a decrease of the stiffness. The effect of addition of bis- and tetrathiol molecules was also considered as tests. These additives lead to increase by 1.5 times the elasticity of gluten-based materials. A multi-scale study (molecular scale by FTIR, macromolecular scale by SEC and macroscopic scale by tensile test, all supported by DTMA analysis) of the structure and properties showed that a predominant conformation with α-helices which is the case of native gluten, leads to a decrease of elasticity. The formation of β-sheets interproteic interactions was identified as the only responsible of elasticity increases of the material. The formation of proteic aggregates with disulfide and thioeter bonds only plays a secondary role. A reaction mechanism highlighting the conditions that ensure the participation of all types of gluten proteins in the gluten network upbuilding is discussed. Two new promising ways of rubber melt and copolymerization by “grafting from” technique were explored and need to be further developed.

No description available.

Eine Studie zur kovalenten Immobilisierung einiger Porphyrinderivate und einer p-Maleimidophenyl-Spezies (p-MP) auf der Si(111)-Oberfläche wird vorgestellt. Hierbei lag der Schwerpunkt auf der Untersuchung des Einflusses von Reaktionsparametern auf die Abscheidung und die Qualität organischer Schichten. Die dünnen Schichten werden mittels nasschemischer Methoden abgeschieden. Die hergestellten Strukturen werden mit einer Vielzahl oberflächensensitiver Messtechniken wie der IR-Ellipsometrie, der XPS-, der SEIRA- und der IR-Reflexionsspektroskopie analysiert. Alle Abscheidungen in dieser Arbeit werden in situ durchgeführt, zusätzlich zu den ex situ Reaktionen, um das Verständnis der Filmwachstums- und Depositionsgeschwindigkeiten zu erhalten. Bei der Untersuchung der Porphyrin-Dünnschichten lag der Fokus auf den synthetische Bedingungen der Materialabscheidung. Die Lösungsreaktionen zeigen, dass die Reaktion mit dem voraktivierten Porphyrinderivat zu einer besseren Ausbeute führt als mit dem in situ aktivierten Porphyrin. Wenn diese Reaktion jedoch unter Verwendung des Schicht-für-Schicht-Abscheidungsprinzips auf die Oberfläche übertragen wird, findet die Reaktion mit den oberflächengebundenen Aminosilanmolekülen mit einer viel langsameren Geschwindigkeit statt als die gleiche Reaktion in Lösung. Es wird ein alternativer Syntheseansatz vorgeschlagen, bei dem das Produkt in Lösung hergestellt und dann auf dem oxidierten Si-Substrat abgeschieden wird. Eine weitere in situ-Studie zum p-MP-Elektropfropfen auf der H-terminierten Si(111)-Oberfläche liefert Details zur Dünnschichtbildung im monolagigen und im sub-monolagigen Bereich. Die spontane Pfropfung von p-MP wird ebenfalls in situ überwacht. Es wurde festgestellt, dass die Bildung einer Monoschicht während der stromlosen Abscheidung länger dauert als bei einer elektrochemischen Abscheidung. Als Alternative zum Radikalmechanismus wird ein kationischer Mechanismus vorgeschlagen. / A study on the covalent immobilization of a couple of porphyrin derivatives and a p-maleimidophenyl species (p-MP) on Si(111) surface is presented to investigate how do reaction parameters influence the deposition and the quality of organic layers. The thin films are deposited with “wet chemistry” methods. The prepared structures are analyzed with a variety of surface sensitive techniques, namely, IR ellipsometry, XPS, SEIRA, and IR reflection spectroscopy. All depositions in this work are conducted in situ, in addition to the ex situ reactions, to gain an understanding of the film growth and deposition rates. The study on porphyrin thin films focused on the synthetic conditions of material deposition. Solution reactions indicate that the reaction with the pre-activated porphyrin derivative leads to a better yield than with the in situ-activated porphyrin. However, when this reaction is transferred to the surface by using the layer-by-layer deposition approach, the reaction with the surface-bound aminosilanes molecules takes place at a much slower rate than the same reaction in solution. An alternative synthetic approach, where the product is prepared in solution and then deposited on the oxidized Si substrate, is proposed. A parallel in situ study on p-MP electrografting on the H-terminated Si(111) surface provides details on the thin film formation in a monolayer and a sub-monolayer regime. The spontaneous grafting of p-MP is also monitored in situ. It was found that the formation of a monolayer during the electroless deposition takes longer than an electrochemical deposition. A cationic mechanism is proposed as an alternative to a radical mechanism.

Blood vessels including the arteries, veins, and capillaries are a critical and indispensable component of various organisms. Some studies estimate that if all the blood vessels present in our body are arranged in line, they would amount to a total length of approximately 60,000 miles. This distance is enough to circle the world two and a half times! In addition to being all pervasive, blood vessels perform certain key functions such as delivery of oxygen and nutrients to various tissues in the body. They also assist in the spread of diseases such as cancer. Therefore, it is important to study vessels from the point of view of tissue engineering applications. In this study, I have adapted the design of an open-source 3D printed device to create channels in Poly (ethylene glycol) Maleimide (PEG-Mal) hydrogels using the subtractive technique. The PEG-Mal hydrogels can be formed in various formulations to mimic the biophysical and biochemical properties of various tissues such as bone marrow, brain, and lung. These channels created within hydrogels can be easily perfused with physiologically relevant flow rates found in blood vessels and capillaries. Additionally, I have also optimized the hydrogel formulations to improve channel reproducibility. It was found that the number of arms of PEG-Mal contributed the most to channel reproducibility with higher success rates of channel formation in 8-arm gels when compared to 4-arm gels. Therefore, this project delineates the formation of simple in vitro channels in hydrogels which combines properties of the tissue specific extracellular matrix with hemodynamics. It is expected that such a system will find potential use in various tissue engineering and disease modeling studies.

Nanoporous organic polymers, including hypercrosslinked polymers (HCPs), covalent organic frameworks (COFs), polymers of intrinsic microporosity (PIMs), and conjugated microporous polymers (CMPs) etc., are considered good candidates for potential gas storage and gas separation applications. Porosities and surface areas of a series of semirigid alternating copolymers, which contained tert-butyl carboxylate-functionalized stilbene or tert-butyl carboxylate-functionalized styrene, and maleic anhydride or tert-butyl carboxylate-functionalized phenyl maleimide, were investigated using nitrogen sorption/desorption isotherms at 77 K and molecular simulations. These alternating copolymers were found to have Brunauer-Emmett-Teller (BET) surface areas in the range of 20-40 m2/g. Surface areas of these alternating copolymers increased as the steric crowding of the polymer backbone increased, which was the result of introducing extra phenyl rings and/or N-phenyl substituent maleimide units. Surface areas were found to increase as the persistence length increased. A series of HCPs containing functionalized stilbene and N-substituted phenyl maleimide were synthesized via free radical suspension polymerization. The incorporation of these functionalized, chain stiffening, Tg enhancing comonomers raised the Tgs of precursor polymers before they were crosslinked. Surface areas of these HCPs, obtained from nitrogen adsorption/desorption isotherms at 77 K, were up to 1058 m2/g. However, the surface areas of these HCPs were systematically lower than the controls. The high rigidity of the polymer backbone, which was the result of incorporating Tg enhancing comonomer, likely affected the chain mobility of the precursor polymer, decreased the efficiency of post-crosslinking reactions, and thus resulted in lower surface areas. Amine-functionalized styrene/stilbene polymers were prepared via free radical polymerization or post-modification. Amine-containing silica-based sorbents were prepared using the impregnation method. Sorption of CO2 by these materials was tested using TGA and compared with control samples. Both high amine content and certain levels of surface area were found to be important for a sorbent to achieve high CO2 uptake. Highest CO2 uptake (12 wt%) under our testing condition in these materials was achieved by an amine-containing silica sorbent. / Ph. D.

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No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2002-12-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main function of the most superficial layer of the epidermis, the Stratum Corneum (SC), is to provide a physical barrier that controls the transepidermal water loss as well as the permeation of another substances in both directions across the skin. The SC is formed by anabolically dead cells, the terminally differentiated corneocyte, and its function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes, termed the cornified cell envelope, and by impeding water diffusion across the SC by mortaring the corneocytes together by layers of skin-specific lipids, essentially ceramide, cholesterol and fatty acid. In this work the cell envelope of the SC was spin labeled with a sulfhydryl-specific nitroxide reagent to investigate the water content effects upon the protein dynamics directly in the intact tissue. A two-state model for the nitroxide side chain described the coexistence of two spectral components in the electron paramagnetic resonance (EPR) spectra. The so-called strongly immobilized component, S, is associated with the EPR signal of a motionally restricted nitroxide fraction having its N-O group hydrogen bonded to protein (rigid structure) while the weakly immobilized component, W, corresponds to the signal provided by the spin labels with higher mobility (~10 times greater) exposed to the aqueous environment. The relative populations between these two mobility states, S and W, are in thermodynamic equilibrium. The standard Gibbs free energy, enthalpy and entropy changes for transferring the nitroxide side chain from the state contacting the solvent, W, to the one contacting protein, S, indicated that the reduction of the SC water content to below ~h 0.69, g H2O per g dry SC, stabilizes the protein interacting state, S. Upon decreasing the SC hydration level below ~h 0.69 the segmental motion of the polypeptide chains and the rotational motion of the spin-labeled side chain were also constrained. To test our methodology in a pure and very well known protein, we also studied the effects of two types of detergents on the bovine serum albumin (BSA). Both detergents, the anionic sodium dodecyl sulfate (SDS) and the zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) increase the mobility of the protein backbone and of the nitroxide side chain. The thermodynamic parameters indicated that these detergents destabilize the protein favoring less compact conformations. This work can also be useful to improve the spectral analysis of site-directed spin labeling, especially for a more quantitative description in terms of thermodynamic parameters. / A camada mais superficial da epiderme, o Estrato Córneo (EC), tem como função principal a formação de uma barreira física que controla a perda de água do corpo bem como a permeação de outras substâncias em ambas as direções da pele. O EC é formado por células anabolicamente mortas, os corneócitos, os quais sofreram diferenciação celular terminal, e sua função é realizada formando uma estrutura de proteínas altamente insolúveis na superfície do corneócito, chamada de envelope celular, e também uma matriz lipídica, essencialmente ceramídios, colesterol e ácidos graxos, que dificultam a difusão da água. Neste trabalho, o EC foi marcado com marcadores de spin específicos para reagir com os grupos sulfidrilas das proteínas, para investigar os efeitos do conteúdo de água na dinâmica de proteínas diretamente no tecido intacto. Um modelo de dois estados para a cadeia lateral do nitróxido descreveu a coexistência de duas componentes espectrais de ressonância paramagnética eletrônica (RPE). A componente denominada fortemente imobilizada (S), surge de uma fração de marcadores com o átomo de oxigênio do nitróxido ligado à proteína (estrutura rígida) enquanto a componente fracamente imobilizada é gerada pelos marcadores com mobilidade mais alta (~10 vezes maior) e expostos ao ambiente aquoso. As populações relativas entre estes dois estados de mobilidade, S e W, estão em equilíbrio termodinâmico. Os parâmetros da termodinâmica: energia livre padrão de Gibbs, entalpia e entropia, envolvidos na transferência da cadeia lateral do nitróxido do estado W, contatando ao solvente, para o estado S, contatando a proteína, indicaram que a redução do conteúdo de água para abaixo de ~0.69g de H2O por g de EC seco, estabiliza o estado S (cadeia lateral do nitróxido dobrada sobre a cadeia principal da proteína). Ao diminuir o nível de hidratação para abaixo de ~ h 0.69 (g H2o/g EC seco) o movimento local da cadeia polipeptídica e o movimento rotacional da cadeia lateral do marcador de spin foram ambos reduzidos. Para testar nossa metodologia em uma proteína pura e bem conhecida, estudamos os efeitos de dois tipos de detergentes sobre a albumina do soro bovino (BSA). Ambos os detergentes, o aniônico dodecil sulfato de sódio (SDS) e o ziteriônico N-hexadecil-N,N-dimetil-3-amônio-1-propanosulfonato (HPS) aumentaram a mobilidade da cadeia principal da proteína e da cadeia lateral do nitróxido. Os parâmetros termodinâmicos indicaram que estes detergentes desestabilizam a proteína favorecendo conformações menos compactas. Os resultados do presente trabalho também podem contribuir para aprimorar a

Maleimide based highly luminescent material Cbz-MI with donor acceptor donor (D-A-D) backbone has been synthesized and characterized. An organic light emitting diode fabricated using this material as emitting layer exhibited EQE of 2.5% in the yellow region of visible spectrum. Due to the small energy gap of materials emitting in this region of spectrum, EQE of OLED is usually limited by various non-radiative decays and high EQE of OLED using this material proves that most of the nonradiative decay pathways have been avoided by the careful design of molecule and device structure. Although Cbz-MI did not show TADF properties, but if tailored with right electron donor along with maleimide as an acceptor, such derivatives may exhibit TADF properties

Capillary electrophoresis mass spectrometry (CE-MS) is a versatile instrumental method for metabolomics, which allows for comprehensive metabolite profiling of volume-limited biological specimens in order to better understand the molecular mechanisms associated with chronic diseases, including an alarming epidemic of obesity worldwide. Multiplexed CE separations enable high-throughput metabolite screening with quality assurance to prevent false discoveries when combined with rigorous method validation, robust experimental designs, complementary statistical methods, and high-resolution tandem mass spectrometry (MS/MS) for unknown metabolite identification. In this thesis, multiplexed CE-MS technology is applied for both targeted and untargeted metabolite profiling of various biological fluids, including covalently bound thiol-protein conjugates, as well as free circulating metabolites in serum and plasma, and excreted/bio-transformed compounds in urine due to complex host-gut microflora co-metabolism. This work was applied to characterize aberrant metabolic responses of obese subjects in response to dietary challenges, and measure the benefits of dietary interventions that reduce adiposity without deleterious muscle loss. Chapter 2 presents, a simple, sensitive yet robust analytical protocol to expand metabolome coverage in CE-MS for the discovery of labile protein thiols in human plasma using a rapid chemical derivatization method based on N-tert-butylmaleimide (NTBM). Chapter 3 describes targeted metabolite profiling of serum and plasma to investigate the differential metabolic responses between healthy and unhealthy obese individuals before and after consumption of a standardized high-caloric meal, respectively. Chapter 4 of this thesis describes an untargeted metabolite profiling strategy for urine using multisegment-injection (MSI)-CE-MS for elucidating the effects of protein supplementation following a short-term dietary weight-loss intervention study. This work revealed six urinary metabolites that were classified as top-ranking treatment response biomarkers useful for discriminating between subjects consuming carbohydrate (control), soy, and whey supplemented diets. In summary, this thesis demonstrated the successful implementation of multiplexed CE-MS technology for biomarker discovery in nutritional-based metabolomic studies as required for more effective treatment and prevention of obesity for innovations in public health. / Thesis / Doctor of Philosophy (PhD)