<p>Currently there is a dearth of analytical techniques for studying protein-ligand interactions on the proteomic scale. Existing techniques, which rely on various calorimetry or spectroscopy methods, are limited in their application to the proteomic scale due to their need for large amounts of pure protein. Recently, several mass spectrometry-based methods have been developed to study protein-ligand interactions. These mass spectrometry-based methods overcome some of the limitations of existing techniques by enabling the analysis of unpurified protein samples. However, the existing mass spectrometry-based methodologies for the analysis of protein-ligand binding interactions are not directly compatible with current mass spectrometry-based proteomics platforms. </p><p>Described here is the development and application of a new technique designed to detect and quantify protein-ligand binding interactions with mass spectrometry-based proteomic platforms. This technique, termed SPROX (Stability of Proteins from Rates of Oxidation), uses an irreversible covalent oxidation labeling reaction to monitor the global unfolding reactions of proteins to measure protein thermodynamic stability. Two variations of the SPROX technique are established here, including one variation that utilizes chemical denaturant to induce protein unfolding and a second variation that utilizes temperature to denature proteins. The SPROX methodology is tested on five proteins including ubiquitin, ribonuclease A, bovine carbonic anhydrase II, cyclophilin A, and calmodulin. Results obtained on these model systems are used to determine the method's ability to measure the thermodynamic parameters associated with each protein's folding/unfolding reaction. Results obtained on calmodulin and cyclophilin A are used to determine the method's ability to quantify the dissociation constants of protein-ligand complexes.</p><p>The primary motivation for the development of the SPROX protocols in this work was to create a protein-ligand binding assay that could be interfaced with conventional mass spectrometry-based platforms. Two specific SPROX protocols, including a label-free approach and an oxygen-16/18 labeling approach, are developed and demonstrated using the thermal SPROX technique to analyze ligand binding in a model four-protein component mixture consisting of ubiquitin, ribonuclease A, bovine carbonic anhydrase, and cyclophilin A. The thermal SPROX technique's ability to detect cyclosporin A binding to cyclophilin A in the context of the model mixture is shown using both labeling approaches. </p><p>An application using the SPROX technique combined with a multi-dimensional protein identification technology (MudPIT)-based proteomics platform is also described. In this application, which utilized an isobaric mass tagging strategy, 325 proteins in a yeast cell lysate are simultaneously assayed for CsA-binding. This study was also used to investigate the protein targets of an already well-studied immunosuppressive drug, cyclosporin A. Two of the ten protein targets identified in this work are known to interact with CsA, one through a direct binding event and one through an indirect binding event. The eight newly discovered protein targets of CsA suggest a molecular basis for post-transplant diabetes mellitus, which is a side effect of CsA in humans.</p> / Dissertation

<p>This work examines the characterization of supramolecular assemblies and, more specifically, host-guest complexes involved in molecular recognition events. The supramolecular assemblies studied take root from metal ion delivery in biological uptake pathways, specifically the delivery of iron to microbial cells. These assemblies are studied in an effort to further understand the nature of molecular recognition events, specifically the nature and strength of interactions between a host and a guest, and possible applications of these systems. </p> <p>The development of a mass spectral method by which to characterize supramolecular assemblies involving the cation binding hosts 18-crown-6, benzo-18-crown-6, dicyclohexano-18-crown-6, and dibenzo-18-crown-6 macrocycles, and the linear ionophore lasalocid with cationic guests, including substituted protonated amines and the iron siderophore ferrioxamine B is presented. Methodology was developed using ESI-MS to successfully quantitate host-guest interactions in binary and complex mixtures. Binding constants were obtained in the range of log Ka = 3 - 5 and correspond to similar systems previously studied in the literature. The studies presented here further our understanding of the molecular recognition events that must occur between a siderophore and a receptor and provide an improved method by which to measure the strength of their interaction. </p> <p>The effects of redox hosts on host-guest complex formation with ferrioxamine B and the characterization of the host-guest complexes formed and the strength of the interactions between them were studied using cyclic voltammetry, ESI-MS, FAB-MS and ITC. A shift in redox potential towards more positive values is observed upon addition of a cationic siderophore guest to a solution of a redox-active para-Wurster's aza crown or mono-substituted Wurster's aza crown macrocycle. Mass spectral evidence indicates the formation of a host-guest complex between the cationic siderophore and the redox host. A redox switch mechanism is proposed, whereby the redox state of the host influences the binding affinity between the host and guest and, consequently, host-guest complex formation. These systems offer a unique means by which to modulate the uptake or release of ionic guests from a cavity by using externally controlled methods and can be applied to selective metal ion compartmentalization. </p> <p>Finally, the application of supramolecular assemblies as a tool in the field of drug delivery is presented. The covalent attachment of an antimalarial drug, artemisinin, by our collaborators to a siderophore produced by M. Tuberculosis, mycobactin, facilitates the subsequent delivery of the drug into the microbial cell by taking advantage of the natural biological iron uptake pathway. Here, the molecular recognition event and supramolecular assembly of interest is that occurring between the siderophore-drug assembly and the microbial receptor. Characterization of the siderophore-drug assembly using cyclic voltammetry shows that there is an interaction between the Fe-mycobactin and artemisinin when these are covalently attached in the form of a conjugate. Increased current output is observed due to an intramolecular electron transfer between the two components. Based on these in vitro data, we propose a redox mechanism by which the drug-siderophore conjugate exhibits a therapeutic effect in vivo.</p> / Dissertation

Thesis (M.SC.) - Carleton University, 2007. / Includes bibliographical references (p. 85-93). Also available in electronic format on the Internet.

Orientador: Frab Norberto Boscolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T03:14:13Z (GMT). No. of bitstreams: 1 Cruz_AdrianaDiboda_M.pdf: 1224328 bytes, checksum: c4ba9884bf5793ca34ee9c248d1bc9ce (MD5) Previous issue date: 2005 / Resumo: A influência da radiação ionizante proveniente de exames radiográficos de diagnóstico ou tratamento radioterápico em materiais como compósitos odontológicos, ionômero de vidro e cerâmica é pouco conhecida. Procurando uma maior compreensão da influência da radiação ionizante no comportamento químico, em escala molecular, dos materiais restauradores estéticos usados na Odontologia, o objetivo neste estudo foi avaliar à ação da radiação aplicada com o aumento de dose e determinar quais possíveis alterações ocorreram na estruturas químicas destes materiais restauradores. Foram confeccionados 25 corpos de prova de cada material, seguindo as recomendações do fabricante e após o período de estabilização dos materiais, foram aplicadas as doses fracionadas de 0,25Gy. Os corpos de prova foram separados em 5 grupos, dentre eles o grupo G1, controle não irradiado, e os grupos G2, G3, G4 e G5, que receberam as doses de 0,25Gy; 0,50Gy; 0,75Gy; e 1,00Gy respectivamente de radiação gama de Cobalto 60. Após um mês da radiação, os corpos de prova foram secos e triturados para a análise espectrométrica. O método usado foi a espectrometria de FTIR com refletância difusa. De acordo com a metodologia empregada e os resultados obtidos, foi possível observar que a radiação ionizante interagiu com todos os materiais odontológicos estudados os quais apresentaram padrão de modificação não linear com aumento da dose de radiação. Vários grupos funcionais se apresentaram susceptíveis, tanto na fração orgânica como na fração inorgânica. Ocorreram alterações na estrutura química de todos materiais irradiados apresentando flutuações quantitativas dos grupos funcionais / Abstract: The influence of the ionizing radiation from diagnosis radiographic exams or radiotherapy treatment in dental materials as resin composite, glass ionomer and ceramic dental is not wide known. Trying a better understanding about the ionizing radiation influence in the chemical behavior, in molecular scale, of the esthetics restoration materials used in the dentistry, our aim with this study was to evaluate the radiation action using a different dose to determine which possible alterations in the chemical structures happened. Twenty-five specimens of each material were performed, following the manufacturer's recommendations, after the materials stabilization period, 0.25Gy fractional doses gamma radiation 60Co were applied. The specimens were separated in 5 groups; group G1, control group not irradiated, and the groups G2, G3, G4 and G5 irradiated with 0.25Gy; 0.50Gy; 0.75Gy; and 1.00Gy respectively. One month after radiation applying, the specimens were dried and triturated for spectrometric analysis. The used method was FTIR spectrometric with diffuse reflectance. According to used methodology and the obtained results, it was possible to observe that the ionizing radiation interacted with all this studied dental materials, which had non lineal pattern of modification with radiation dose increase. Several functional groups are susceptible, as well as in the organic fraction and in the inorganic fraction. All the irradiated materials had alterations in the chemical structure presenting quantitative fluctuation of the functional groups / Mestrado / Radiologia Odontologica / Mestre em Radiologia Odontológica

Doctor of Philosophy / Department of Chemistry / Peter M. Sherwood / This work is a detailed study of the interaction between two phosphorous-containing acids and the metals chromium and 316L stainless steel. The objective of this work is to investigate the formation of unique thin films on the two metals and to probe the surface chemistry of these films through the use of core level and valence band X-ray photoelectron spectroscopy (XPS). Chromium forms a wide array of oxides and can exist at several valencies. Valence band XPS is used in conjunction with band structure and multiple scattered wave X[alpha] calculations to distinguish which states are present in the resultant films. Both 99.99% chromium and 316L stainless steel foils were treated with orthophosphoric acid and 1-hydroxyethylidene-1,1-diphosphonic acid, otherwise known as etidronic acid. Two methods developed in the Sherwood research laboratory for forming oxide-free films on metal surfaces are utilized in this work. Core level XPS results did not provide sufficient information to draw conclusions regarding the products formed in the reactions. The valence band results showed clear evidence of multiple forms of phosphates forming on the metal surfaces as evidenced by the subtle differences in separation between the phosphorous 3p and 3s peaks as well as differences in separation between the O2s and phosphorous 3s peaks. The Valence Band XPS results were interpreted by X-[alpha] cluster and band structure calculations. Films formed on chromium foil from the orthophosphoric acid were found to be condensed phosphates that are stable in air. Etidronic acid formed very thin phosphate films on chromium with both treatment methods as well as on 316L stainless steel when the bench top method was applied. Treatment of etched 316L steel in the anaerobic cell generated an etidronate film. This sample was the only etidronate film formed, all other etidronate-based films were generated from disassembled portions of the etidronate ion to form phosphate films.

Master of Science / Department of Chemistry / Christopher T. Culbertson / The field of microfluidic devices has being developed quickly. It is aimed at integration of many chemical functions in a single chip, such as sample pretreatment, preconcentration, separation and detection, which provides a number of advantages including simplicity, automation, reduced analysis time, decrease in amount of samples and reduced formation of waste. Its potential applications have been conducted in the fields such as biotechnology, pharmaceuticals, life sciences, public health, agriculture and related areas. Membrane technology has been applied in analytical chemistry for many years and has won substantial growth in microfluidics over the past 10 years. Membrane is used to control transfer of some kinds of species, which can be employed for sample concentration, sample preparation, sample filtration and microreactors and so on. Sol-gel process, which usually involves catalytic hydrolysis of sol-gel precursor(s) and catalytic polycondensation of the hydrolyzed products and other sol-gel-active components present in the reaction medium to form a macromolecular network structure, is one of the most suitable methods for membrane fabrication. In this work, titanium membrane was fabricated inside glass microchips using the precursor of titanium isopropoxide. The resulted membranes demonstrated the excellent preconcentration effect. Followed separation and detection were also achieved. CE has been highly accepted as an efficient separation technique for qualitative and quantitative determination, which is performed using a narrow-bore capillary tube. It offers advantages of simplicity, high resolution separation, and minimal cost in terms of analysis time and sample consumption. In this work, protein separations were carried out by CE. Laser-induced fluorescence was used as detection. On-column fluorescence labeling using a fluorogenic labeling reagent was made. Under suitable experimental conditions, an excellent separation performance with about 1.4 million theoretical plate numbers was achieved.

L’adénocarcinome pancréatique ductal (PDAC) est une des tumeurs solides les plus malignes retrouvées dans la population humaine actuelle. Plusieurs chercheurs s’intéressent donc à trouver un nouveau traitement efficace pour ce type de cancer. Les biguanides, souvent utilisés comme antidiabétiques, intéressent de plus en plus les chercheurs par la découverte de leurs propriétés antiprolifératives. Dans notre groupe, une variété de nouveaux biguanides a été synthétisée et étudiée pour leurs propriétés antiprolifératives des cellules cancéreuses du pancréas. Nous avons identifié un biguanide avec une chaîne de phényléthynylbenzyle, appelé composé A, comme un composé de choix par son activité 800 fois plus élevée que celle de la metformine et 10 fois plus élevée que celle de la phenformine, deux biguanides commercialement disponibles. Des études in vivo ont été effectuées en greffant des cellules PDAC humaines dans l’espace sous-cutané de souris et laissées croître pendant 28 jours jusqu’à une taille de 100 mm3. Différents groupes de souris ont reçu des doses quotidiennes de 50 mg/kg de metformine, phenformine ou du composé A. Par la suite, les souris ont été sacrifiées afin de recueillir les organes et les tumeurs pour des analyses par spectrométrie de masse MALDI-TOF. Le développement d’une méthode d’analyse par MALDI-TOF a permis de localiser et quantifier les biguanides dans les organes et tumeurs. Différents paramètres, tels que le choix de matrice et la méthode de dépôt, ont été optimisés afin d’obtenir des courbes d’étalonnage par IMS des trois composés biguanides. Une accumulation du composé A est observée dans le foie et le rein. Avec une régression linéaire, les concentrations approximatives du composé A dans le rein et le foie sont de 16,8 µg/mL et 4,1 µg/mL respectivement. De plus, pour en apprendre davantage sur le mécanisme d’action des biguanides dans le traitement du cancer, une étude différentielle d’expression de certaines biomolécules a été effectuée sur les foies des différents groupes de souris traitées. Des différences d’expression de certaines biomolécules ont été observées par rapport aux foies des animaux non-traités, notamment pour le signal à m/z 558 qui montre une grande abondance de ce phospholipide seulement dans les foies traités au composé A. Bref, le mécanisme d’action des biguanides dans le traitement du cancer demeure à l’étude pour de futurs travaux et développements. / The development of new therapies against pancreatic ductal adenocarcinoma (PDAC), one of the most malignant and deadly tumors, has become of great interest for finding effective treatment. Many research groups have studied different types of biguanides in cancer treatment which inspired our group to synthesize a library of biguanide analogs. These new compounds can inhibit the tumor growth and show a great selectivity. We identified a biguanide with a phenylethynylbenzyl side chain as our hit compound, designated as compound A, which is 800 times more active than metformin and 10 times more active than phenformin, two commercial biguanide compounds used as antidiabetics. In vivo studies have been performed where human PDAC cells have been grafted in the subcutaneous space of mice and let to grow for 28 days to about 100 mm3. Different groups of mice were exposed to daily doses of 50mg/kg of metformin, phenformin or compound A. The tumors and organs of the treated mice were recovered to analyze their mechanism of action by MALDI-TOF mass spectrometry. A method of analysis by MALDI-TOF was developed in order to locate and quantify the biguanide compounds in the treated organs and tumors. Various parameters, such as the choice of matrix and the deposition method were optimized to obtain IMS calibration curves for each biguanide compound. The analyzed organs and tumors showed accumulation of compound A in the liver and kidney. With linear regression, this semi-quantitative method allowed to determine a concentration of 16.8 µg/mL in the kidney and 4.1 µg/mL in the liver. Another objective to understand the mechanism of action of biguanides in cancer therapy is to detect molecular patterns of biomolecules that correlate with the diagnostic, the prognostic and to the response of the therapy. The livers from mice treated with compound A showed differences of expression of certain phospholipids compared to the other livers, namely at m/z 558. In short, the mechanism of action of biguanides in cancer therapy is still a work in progress for future developments.

Modern three-dimensional integrated circuit design is rapidly evolving to more complex architecture. With continuous downscaling of devices, there is a pressing need for metrology tool development for rapid but efficient process and material characterization. In this dissertation work, application of a novel multiple internal reflection infrared spectroscopy metrology is discussed in various semiconductor fabrication process development. Firstly, chemical bonding structure of thin fluorocarbon polymer film deposited on patterned nanostructures was elucidated. Different functional groups were identified by specific derivatization reactions and model bonding configuration was proposed for the first time. In a continued effort, wet removal of these fluorocarbon polymer was investigated in presence of UV light. Mechanistic hypothesis for UV-assisted enhanced polymer cleaning efficiency was put forward supported by detailed theoretical consideration and experimental evidence. In another endeavor, plasma-induced damage to porous low-dielectric constant interlayer dielectric material was studied. Both qualitative and quantitative analyses of dielectric degradation in terms of increased silanol content and carbon depletion provided directions towards less aggressive plasma etch and strip process development. Infrared spectroscopy metrology was also utilized in surface functionalization evaluation of very thin organic films deposited by wet and dry chemistries. Palladium binding by surface amine groups was examined in plasma-polymerized amorphous hydrocarbon films and in self-assembled aminosilane thin films. Comparison of amine concentration under different deposition conditions guided effective process optimization. A time- and cost-effective method such as current FTIR metrology that provides in-depth chemical information about thin films, surfaces, interfaces and bulk layers can be increasingly valuable as critical dimensions continue to scale down and subtle process variances begin to have a significant impact on device performance.

Artificial photosynthesis, for the purpose of converting solar energy into fuel, is one of the most viable and promising alternative approaches to solve the current global energy and environmental issues. Among the challenges faced in artificial photosynthesis is in building photosystems that can effectively and efficiently perform light absorption and charge separation in broad-band capturing donor-acceptor systems. While having a broad-band capturing antenna system that can harness incoming photons is crucial, another equally important task is to successfully couple the antenna system, while maintaining its optical properties, to an energy or electron acceptor which serves as the reaction center for the generation of charged species of useful potential energy. The stored potential energy will be utilized in different applications such as driving electrons in solar cells or in splitting water for the generation of fuel. Hence, the particular endeavor of this thesis is to study and synthesize molecular/supramolecular systems with wide-band capturing capabilities to generate long-lived charge separated states. The sensitizer used in building these systems in the present study is 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, for short, BF2 chelated Azaboron dipyrromenthene or AzaBODIPY. A handful of novel donor-acceptor systems based on AzaBODIPY have been successfully designed, synthesized and their photochemistry have been investigated using various techniques. In these systems, Azabodipy has been covalently attached to several donors like porphyrin, bodipy, subphthalocyanine, phenothiazine, ferrocene, bithiophene and effectively coupled to an electron acceptor, C60. These systems have been fully characterized by NMR, Mass, optical absorption and emission, X-ray crystallographic, computational, electrochemical, and photochemical studies. It has been possible to demonstrate occurrence of efficient electron and energy transfer events and long-lived charge separated states upon photoexcitation in these model compounds. By changing the arrangements of the donor and acceptor entities, it has also been possible to show directional, through-space and through-bond electron transfer processes. The present study brings out the importance of utilizing near-IR sensitizers in building solar energy harvesting model systems.

The increasingly complex world of illicit chemistry has created a need for rapid, selective means of determining the threat posed by new drugs as they are encountered by law enforcement personnel. To streamline this process, the entirety of the problem, from the production of illicit drugs all the way to the final analysis have been investigated. A series of N-alkylated phenethylamine analogues were synthesized in a shotgun method and subjected to direct-infusion analysis. A range of products were detected without the need for time-consuming purification steps, which was extended to novel pharmacological and receptor-binding assays where mass spectrometry is used as a detector. This direct-infusion technique was also applied to studies of methamphetamine and fentanyl production to preemptively determine improvements to common reaction conditions and explore the origins of common impurities. The ability to utilize these rapid techniques directly from the fume hood has also been critically reviewed to highlight gaps in current research and opportunities for improvement. When combined, these studies seek to provide a means for rapid, simplified analysis of illicit drugs to improve the quality of data and dramatically increase throughput.