The metabolism of amino acids produces important chemical signaling molecules called neurotransmitters, which are responsible for carrying out important actions within the human body. There are approximately one hundred identified neurotransmitters. Neurotransmitter study is important due to their involvement in biological, physiological, pharmacological, and pathological functions. Commonly employed methods for neurotransmitter detection are mainly based upon microdialysis. However, the methods suffer from disadvantages. Microdialysis fails to determine the absolute concentration of analytes and therefore requires it to be tied in with an analytical technique such as high performance liquid chromatography or capillary electrophoresis. Although high performance liquid chromatography is the most powerful analytical technique to date, it necessitates high maintenance and suffers from poor temporal resolution. While capillary electrophoresis affords more rapid separations than high performance liquid chromatography, it suffers from poor concentration limits of detection and requires large sample dilutions of highly conductive samples, such as biological fluids. Consequently, research is focused on detection of various amino acids and neurotransmitters employing novel analytical techniques along with traditional capillary electrophoresis. First, a method was developed using traditional capillary electrophoresis with laser induced fluorescence detection to detect two major excitatory neurotransmitters, glutamate and aspartate in planaria. The method was later applied to detect several biogenic amines using micellar electrokinetic chromatography with laser induced fluorescence detection in planaria to study the effect of feeding on the levels of biogenic amines within individual planaria homogenates. The concentration sensitivity issue of capillary electrophoresis led to the use of a new method for sensitive neurotransmitter measurements, gradient elution isotachophoresis. Gradient elution isotachophoresis is an efficient capillary-based enrichment and separation technique based on balancing hydrodynamic counter-flow against electrophoresis. Enrichment is achieved with the aid of high concentrations of leading electrolyte in the counter-flow solution that creates an ionic interface near the capillary inlet. Discrete electrolyte spacers or carrier ampholyte mixtures are used to separate analyte zones. The method was applied to the enrichment and separation of physiologically relevant concentrations of aspartate and glutamate labeled with dansyl chloride, phenyl isothiocyanate, or carboxyfluorescein, succinimidyl ester in artificial cerebrospinal fluid using ultraviolet absorbance detection. Finally, gradient elution isotachophoresis was combined with capillary zone electrophoresis to eliminate the use of spacers and provide rapid separations and enrichment. The technique was applied for the detection of biogenic amines in a glass microfluidic device. / Chemistry

This dissertation research work focuses on the investigation of novel donor-acceptor systems elucidating their photochemical properties, anion binding, and their potential application in the development of artificial photosynthetic systems. The explored systems are based on oxoporphyrinogen (OxPs), porphyrins, fullerene, and boron dipyrromethene (BODIPY) based donor-acceptor systems. The photochemical properties of novel molecular systems were elucidated using UV-vis spectroscopy, fluorescence spectroscopy, electrochemical methods, computational calculations, and ultrafast transient absorption spectroscopy. A novel BODIPY-oxoporphyrinogen dyad which is able to bind with fluoride anion promoting the excited state ultrafast electron and energy transfer events mimicking the primary events in natural photosynthesis was introduced. Further, self-assembly of supramolecular complexes based on oxoporphyrinogens, fullerene, and different zinc porphyrin dimers was explored. The formed self-assembled complexes have shown photoinduced electron transfer. A novel push-pull supramolecular construct based on the spiro-locked N-heterocycle-fused zinc porphyrin was studied. The excited state charge separation and stabilization of this push-pull system was enhanced by the complexation with fluoride anion. Also, the effect of BODIPY functionalization and linkers on the electron transfer properties of a series of carbazole–BODIPY and phenothiazine-BODIPY dyads were investigated. These findings are important to develop advanced and efficient BODIPY-based donor-acceptor systems for efficient light harvesting applications. The entire study aims to expand our understanding of these systems and contribute towards the advancement of sustainable energy technologies.

Cells have been found to have an inherent heterogeneity that has led to an increase in the development of single-cell analysis methods to characterize the extent of heterogeneity that can be found in seemingly identical cells. With an understanding of normal cellular variability, the identification of disease induced cellular changes, known as biomarkers, may become more apparent and readily detectable. Biomarker discovery in single-cells is challenging and needs to focus on molecules that are abundant in cells. Lipids are widely abundant in cells and play active roles in cellular signaling, energy metabolism, and are the main component of cellular membranes. The regulation of lipid metabolism is often disrupted or lost during disease progression, especially in cancer, making them ideal candidates as biomarkers. Challenges exist in the analysis of lipids beyond those of single-cell analysis. Lipid extraction solvents must be compatible with the lipid or lipids of interest. Many lipids are isobaric making mass spectrometry analysis difficult without separations. Single-cell extractions using nanomanipulation coupled to mass spectrometry has shown to be an excellent method for lipid analysis of tissues and cell cultures. Extraction solvents are tunable for specific lipid classes, nanomanipulation prevents damage to neighboring cells, and lipid separations are possible through phase dispersion. The most important aspect of single-cell analysis is that it uncovers the extent of cellular heterogeneity that exists among cellular populations that remains undetected during averaged sampling.

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.

The regulations of many food products in the United States have been made and followed very well but unfortunately some products are not put under such rigorous standards as others. This leads to products being sold, that are thought to be healthy, but in reality contain unknown ingredients that may be hazardous to the consumers. With the use of several instrumentations and techniques the detection, characterization and identification of these unknown contaminates can be determined. Both the AZ-100 and the TE2000 inverted microscope were used for visual characterizations, image collection and to help guide the extraction. Direct analyte-probed nanoextraction (DAPNe) technique and nanospray ionization mass spectrometry (NSI-MS) was the technique used for examination and identification of all adulterants. A Raman imaging technique was than introduced and has proven to be a rapid, non-destructive and distinctive way to localize a specific adulterant. By compiling these techniques then applying them to the FDA supplied test samples three major adulterants were detected and identified.

L’imagerie par spectrométrie de masse (IMS) est une technique en pleine expansion et utilisée dans beaucoup d’études effectuées sur des systèmes biologiques tels que la corrélation entre l’expression moléculaire et l’état de santé d’un tissu et pour étudier la biologie du développement. Cependant, plus particulièrement lors de l’analyse de protéines, seulement les molécules les plus abondantes et/ou les plus facilement ionisables seront détectées. L’une des approches utilisées pour éviter cette limitation est de transférer les protéines de manière sélective à partir d’une section tissulaire mince vers une surface fonctionnalisée tout en maintenant leur organisation spatiale. Dans ce cas, seulement les protéines possédant une affinité pour la surface seront alors retenues alors que les autres seront éliminées. Donc, la nature chimique de cette surface est critique. Les travaux de recherches présentés dans ce mémoire portent sur le développement d’une méthode de transfert des protéines d’une section tissulaire vers une surface composée de nitrocellulose. Cette méthode utilise un système permettant d’effectuer le transfert sans contact physique direct entre les surfaces. De plus, lors du transfert, une pression physique est appliquée. Dans une première approche, la méthode développée a été appliquée en utilisant une section de rein de souris comme échantillon modèle. Des sections sérielles ont été collectées, soit pour être colorées à l’aide d’hématoxyline et d’éosine (H&E) afin de démontrer la régiospécificité du transfert, soit pour être analysées directement par IMS afin de déterminer si les protéines détectées après transfert sont également détecter dans les sections analysées directement. Les résultats obtenus ont démontré qu’un sous-ensemble de protéines a été transféré tout en conservant leur position spatiale initiale dans les sections. Certains signaux observés pour les protéines transférées sont uniques et/ou sont nettement mieux détectés que lors de l’analyse directe d’une section. / Imaging mass spectrometry (IMS) is a technique in full expansion that is used in a large range of studies such as the correlation between molecular expression and the health status of a tissue and developmental biology. A common limitation of the technology is that only the more abundant and/or more easily ionisable molecules are usually detected, in particular in protein analysis. One of the methods used to alleviate this limitation is the direct specific transfer of proteins from a tissue section to a functionalized surface with high spatial fidelity. In this case, only proteins with an affinity for the surface will be retained whereas others will be removed. The chemical nature of the surface is therefore critical. The research work presented in this document proposes a high spatial fidelity transfer method for proteins from a tissue section onto a nitrocellulose surface. The method uses a homebuilt apparatus that allows the transfer process to be done without any direct physical contact between the tissue section and the transfer surface while still using physical pressure to help protein migration. In subsequent work, the developed method was used to transfer proteins from a mouse kidney section onto the nitrocellulose surface. Serials sections were also collected either to be colored with hematoxylin and eosin (H&E) to assess the high spatial fidelity of the transfer process, or to be directly analyzed as a control sample to access the different signals detected after transfer. Results showed a high spatial fidelity transfer of a subset of proteins. Some of the detected transferred proteins were not observed after direct tissue analysis and/or showed an increase in sensitivity.

À cause de leurs propriétés antibactériennes, les nanoparticules d’argent sont couramment utilisées dans un grand nombre de produits tels les tissus, les savons et les produits médicaux. Dans cette industrie en pleine croissance, ces nanoparticules sont produites en grandes quantités et s’accumuleront éventuellement dans l’environnement. Pour comprendre le destin, le transport et la biodisponibilité des nanomatériaux, il est essentiel de comprendre leurs propriétés physicochimiques. Entre autres, il est particulièrement important de quantifier la dissolution des nanoparticules à l’aide de mesures de spéciation chimique. En effet, l’objectif de cette recherche est de déterminer la spéciation chimique des nanoparticules d’argent dans différents sols. Pour y parvenir, différentes concentrations de nanoparticules d’argent ont été incorporées dans un sol et après un certain laps de temps, la forme ionique a été mesurée à l’aide d’une électrode sélective d’argent tandis que l’argent total est mesuré par absorption atomique ou par ICP-MS. L’analyse de la spéciation dans trois sols différents révèle que les caractéristiques des sols influencent grandement la spéciation chimique, plus particulièrement la quantité de matière organique ainsi que le pH du sol. Ainsi, la tendance des résultats semble indiquer que plus un sol est acide, il y aura plus d’ions argent libres tandis que la matière organique adsorbe fortement les ions argent les rendant ainsi moins disponibles en solution. L’observation de la spéciation chimique à long terme indique aussi que les nanoparticules tendent à éventuellement se dissocier et ainsi émettre un plus grand nombre d’ions dans l’environnement. Ces résultats ont des implications importantes dans la détermination des risques environnementaux des nanoparticules métalliques. / Because of their antibacterial properties, silver nanoparticles are widely used in common items such as textiles, soaps and medical products. This practice has shown a drastic expansion during the last years thus leading to potential contamination of the environment by nanoparticles. To understand fate, transport and bioavailabity of nanoparticles, it is important to understand their physicochemical properties. More specifically, it is essential to quantify the dissolution of nanoparticles with chemical speciation measurements. The aim of this study is to quantify the speciation of silver nanoparticles in different soils. Different concentrations of silver nanoparticles have been injected in soil and after a specific time, the ionic form was measured with a silver specific electrode while total silver was quantified by atomic absorption or ICP-MS. Chemical speciation measurements in three different soils indicate that a soil’s properties has a large influence on the fate of silver nanoparticles, especially it’s pH and organic matter content. Results show that if a soil is acidic, it will lead to the release of more free silver ions while organic matter tends to adsorb ions making them less available. Over a six month period, nanoparticles seem to fix rapidly to soil solids but also seem to dissociate or oxidise over the months, leading to a greater amount of potentially bioavailable ions. These results have important implications to the determination of environmental risks of metal nanoparticles.

L’explosion de la nanotechnologie a permis l’intégration d’une multitude de nanoparticules dans des produits de consommation. Les nanoparticules d’argent (nAg) sont les plus utilisées à ces fins, selon les derniers recensements disponibles. La plupart des études toxicologiques, à ce jour, ont fait état de l’implication très évidente de l’ion Ag+ dans la toxicité aigüe des nAg; cependant, quelques études ont mis en évidence des effets toxicologiques dus aux nAg. Il y a un certain consensus à propos d’un risque de contamination des eaux douces via leur rejet par les effluents des réseaux d’aqueducs. Puisque les concentrations en Ag+ sont généralement très faibles dans les eaux douces (de l’ordre du pg L-1), de par la formation de complexes non-labiles avec des thiols (organiques et inorganiques) et des sulfures, la toxicité inhérente aux nAg pourrait ne pas être négligeable- comparativement aux tests en laboratoires. Cette étude s’intéressait donc aux mécanismes de bioaccumulation d’argent par l’algue verte C. reinhardtii suite à l’exposition à des nAg de 5 nm (enrobage d’acide polyacrylique). La bioaccumulation d’argent pour l’exposition à Ag+ servait de point de comparaison; également, les abondances de l’ARNm de l’isocitrate lyase 1 (ICL1) et de l’ARNm de Copper Transporter 2 (CTR2) étaient mesurées comme témoins biologiques de la bioaccumulation de Ag+. Les expériences ont été menées en présence d’un tampon organique (NaHEPES, 2 x 10-2 M; Ca2+, 5x 10-5 M) à pH de 7,00. Pour des expositions à temps fixe de 2 heures, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+; cependant, il n’y avait pas de différence d’abondance des ARNm de ICL1 et de CTR2 entre nAg et Ag+. D’un autre côté, pour une exposition à temps variables, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+ et une augmentation de l’abondance de l’ARNm de ICL1 était notée pour nAg. Cependant, il n’y avait aucune différence significative au niveau de l’abondance de l’ARNm de CTR2 entre nAg et une solution équivalente en Ag+. L’ajout d’un fort ligand organique (L-Cystéine; log K= 11,5) à une solution de nAg en diminuait radicalement la bioaccumulation d’argent par rapport à nAg-sans ajout de ligand. Par contre, l’abondance des ARNm de ICL1 et de CTR2 étaient stimulées significativement par rapport à une solution contrôle non-exposée à nAg, ni à Ag+. Les résultats suggéraient fortement que les nAg généraient des ions Ag+ au contact de C. reinhardtii. / The recent developments in nanotechnology have given rise to a new and increasing economical market where nanoparticles are at the forefront. Recent inventories of the nanoparticles-containing products have shown that silver nanoparticle- containing products are the most frequently used consumer nanomaterial. Due to the fear of a large scale contamination-and even pollution- of the aquatic environment from silver nanoparticles (nAg), studies have been conducted to assess their toxicities, which, in many cases, have been found to be mediated by the concomitant presence of Ag+. Notably, few studies have found evidence of toxicity due to the nAg, per se. Since numerous non-labile complexes are formed with Ag+ in freshwaters- especially with thiols and sulfides-, nAg toxicity might be more relevant in comparison to laboratory tests where the Ag+ tends to dominate toxicity studies. Therefore, this study investigated the mechanisms underlying silver bioaccumulation by the green alga, C. reinhardtii upon exposure to solutions of nAg (nominal size of 5 nm; poly-acrylate coating). Silver bioaccumulation upon exposures to the free ion alone served for comparison. In parallel, the abundance of two mRNAs- ICL1 and CTR2- were used to better understand the mechanisms underlying the bioaccumulation of Ag+ (and potentially nAg). The experiments were conducted in pH buffered solutions (NaHEPES, 2 x 10-2 M; Ca2+, 5x 10-5 M) at pH 7.00. For 2-hour exposures, the silver bioaccumulation for solutions of nAg exceeded what was expected from their Ag+ content only; however, no differences were noticed in the abundance of the expression of ICL1 and CTR2. For variable time exposures, the silver bioaccumulation for solutions of nAg exceeded what was expected from their Ag+ content only. Moreover, the expression of ICL1 was significantly higher for nAg than what was expected based upon an exposure to Ag+ only. When exposed to nAg, expression levels of CTR2 could be predicted from levels based solely on the Ag+ concentrations. The addition of a large excess of L-Cysteine, which is a very strong silver ligand (log K =11.5), to a nAg solution largely decreased silver bioaccumulation, however, bioaccumulation remained significant and the expression of both ICL1 and CTR2 were significantly higher than that of the control solutions (without Ag+). The results strongly suggest that nAg generated Ag+ ions when in contact with C. reinhardtii and that the nAg released to freshwaters might exert its toxicity through organism-contact-dependant release of Ag+.

No description available.

<p>Many of the biological roles of proteins are modulated through protein-ligand interactions, making proteins important targets for drug therapies and diagnostic imaging probes. The discovery of novel ligands for a protein of interest often relies on the use of high throughput screening (HTS) technologies designed to detect protein-ligand binding. The basis of one such technology is a recently reported mass spectrometry-based assay termed SUPREX (stability of unpurified proteins from rates of H/D exchange). SUPREX is a technique that uses H/D exchange and MALDI-mass spectrometry for the measurement of protein stabilities and protein-ligand binding affinities. The single-point SUPREX assay is an abbreviated form of SUPREX that is capable of detecting protein-ligand interactions in a high throughput manner by exploiting the change in protein stability that occurs upon ligand binding.</p><p>This work is focused on the development and application of high throughput SUPREX protocols for the detection of protein-ligand binding. The first step in this process was to explore the scope of SUPREX for the analysis of non-two-state proteins to determine whether this large subset of proteins would be amenable to SUPREX analyses. Studies conducted on two model proteins, Bcl-xL and alanine:glyoxylate aminotransferase, indicate that SUPREX can be used to detect and quantify the strength of protein-ligand binding interactions in non-two-state proteins.</p><p>The throughput and efficiency of a high throughput SUPREX protocol (i.e., single-point SUPREX) was also evaluated in this work. As part of this evaluation, cyclophilin A, a protein target of diagnostic and therapeutic significance, was screened against the 880-member Prestwick Chemical Library to identify novel ligands that might be useful as therapeutics or imaging agents for lung cancer. This screening not only established the analytical parameters of the assay, but it revealed a limitation of the technique: the efficiency of the assay is highly dependent on the precision of each mass measurement, which generally decreases as protein size increases. </p><p>To overcome this limitation and improve the efficiency and generality of the assay, a new SUPREX protocol was developed that incorporated a protease digestion step into the single-point SUPREX protocol. This new protocol was tested on two model proteins, cyclophilin A and alanine:glyoxylate aminotransferase, and was found to result in a significant improvement in the efficiency of the SUPREX assay in HTS applications. This body of work resulted in advancements in the use of SUPREX for high throughput applications and laid the groundwork for future HTS campaigns on target proteins of medical significance.</p> / Dissertation