Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar. / doctoral

After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.

A study was made to evaluate how current measurementscould help to determine how particles from fish farms are dispersed. During sixweeks of the summer 2012, two different current measurements were conducted.One of them consisted of measurements each fifth minute at 5 m and 15 m atthree locations for 14-16 days each. The other consisted of profilemeasurements at each location at four times during the period of six weeks. Forthe current measurements two instruments (model RCM 9) were used. A two weekmeasurement can give enough data to make an evaluation of how the currents at alocation will transport litter from a fish farm. A longer probing time wouldhowever be desirable since the currents along the northern east coast of Swedenare mainly driven by factors that are changing during the year such as airpressure, temperature, precipitation etc. Water current measurements can be ofgreat help both when determining how particles from a fish farm is dispersedand also for governing authorities in the processes of both allowing new fishfarms and also when supervision of fish farms is needed.

Fatty acid profiling provides information on dietary intakes and an understanding of lipid metabolism. High throughput techniques such as fingertip prick (FTP) sampling has gained popularity in recent years as a simplified method for basic research, and could be further used to assess disease risk in the population, and other similar high-throughput techniques have the potential to assist in the monitoring and labeling of fatty acids in the food supply. With the advancement of high-throughput sample analysis techniques, a more complete understanding of storage stability is required as a larger volume of samples are produced with equal amounts of time to analyze them. Energy-assisted analysis techniques have the potential to help ameliorate some of these issues. Presently, FTP blood, whole blood and salmon storage stability is assessed under various storage conditions, and both microwave-assisted direct transesterification and indirect ultrasound-assisted extraction techniques are assessed. It is determined that storage of FTP blood and whole blood samples at -20°C results in significant and nearly complete highly unsaturated fatty acid (HUFA) degradation compared to all other temperatures examined. This degradation is determined to be the result of hemolysis and subsequent iron release from erythrocytes initiating fatty acid peroxidation reactions. Direct transesterification of FTP blood is reduced from as long as three hours to one minute with microwave-assisted energy and fatty acid extraction from ground flaxseed is reduced to 40 minutes from as long as 24 hours without compromising fatty acid profiles. Results of the current study provides insight into the storage stability of food sample and blood samples collected via high-throughput techniques, and provides support for the utilization of further high-throughput energy-assisted analytical methods that can help to minimize the potentially detrimental effects that long-term storage can have on fatty acid profiles.

A method for the determination of platinum group elements (PGEs) in natural tree samples was developed. An alumina column in-line with inductively coupled plasma mass spectrometry (ICP-MS) achieves the separation of interferents as well as pre-concentration of the analytes. The application of this proposed method on tree top samples displayed an effective separation of Ru, Rh, Re, Pd, Ir and Pt from the interferents, Ni, Cu and Zn for quantitative analysis of the analytes. The concentration data was compared to ICP-HRMS data and while it was difficult to determine whether the concentrations were in agreement or not, as both methods have a large degree of error. However, both methods displayed elevated concentrations of PGEs in areas over geological conductors in Rock Lake, Manitoba. This proposed method offers distinct advantages over previous on-line methods, as it is extended to include multiple PGEs as well as reduces sample consumption to a more suitable volume for natural samples. While the detection limit is higher than previous methods due to the lowered sample volume, it is still lower than the detection limits reported in commercial laboratories. / Thesis (Master, Chemistry) -- Queen's University, 2008-01-30 19:40:54.673 / Anglo American

Volvo Cars Corporation (VCC) is devoted to Lean and Six Sigma, and has lately considered an imple-mentation of Design for Manufacturing and Assembly (DFMA) with the purpose of improving their product design process and production. The goal for this project work is therefore to investigate if DFMA is a beneficial method for VCC to use, how DFMA can be used and suggest a DFMA method. A literature study is performed as a starting point to understand DFMA methods and its possibilities. Scientific articles, technical books and online sources is used during the study in order to gather in-formation regarding DFMA implementations, application areas, approaches and potential risks that need to be eliminated for a successful implementation. In order to map the product development and manufacturing processes of VCC interviews are per-formed. The interviews also gathered information regarding what VCC employees thinks of DFMA and how they wish to use it. This information is compiled into a demand specification where the de-mands are weighted after importance by the interviewees. After the pre-study, three idea generation sessions is performed with three different focus groups. The generated ideas are then evaluated and classified. The remaining ideas are classified with the purpose of enabling the possibility to use a morphological chart to build whole concepts from the single ideas. Three concepts are chosen amongst the generated concepts. These concepts are then evaluated against the weighted demand specification. The concept considered most suitable is further devel-oped which resulted in a proposed DFMA method for VCC. A case study on a product is performed in order to communicate, test and evaluate the final DFMA method. The project is rounded off with discussions regarding DFMA and the suggested method from sustain-ability point of view and how to motivate usage. All results and discussions from the project are handed over to the company, enabling further research on a possible implementation of DFMA in the organization. / Volvo Cars Corporation arbetar hängivet efter metoderna Lean och Sex Sigma och har avsikt att im-plementera Design for Manufacturing and Assembly (DFMA) i syfte att förbättra deras produkt- och produktutvecklingsprocess. Målet med detta arbete är att undersöka om DFMA är en värdefull me-tod för VCC att använda och hur metoden kan tänkas användas och implementeras inom organisat-ionen. Som utgångspunkt för att förstå DFMA metoden och dess möjligheter genomförs en litteraturstudie. Under studien granskades aktuella vetenskapliga artiklar, tekniska böcker och webbkällor i syfte att samla information om DFMA-implementeringar, användningsområden, tillvägagångsätt och potenti-ella risker som behövs elimineras för en lyckad implementation av metoden. Vidare utförs intervjuer i syfte att kartlägga produktutvecklings- och tillverkningsprocessen samt samla information om hur anställda ställer sig till, och önskar använda DFMA. Denna information sammanställs sedan till en kravspecifikation där kraven i sin tur viktas av intervjuobjekten efter hur viktiga de anses vara. Tre idégenereringssessioner utförs därefter med tre olika fokusgrupper. Dessa idéer gallras sedan ut och klassificeras för att sedan kombineras med hjälp av en morfologisk tabell i syfte att bygga kon-cept av de enskilda idéerna. Tre koncept väljs ut bland de genererade koncepten. Dessa koncept utvärderas sedan mot varandra med kravspecifikationen som bedömningsskala. Det koncept som bedöms som mest lämpligt vidare-utvecklas och en föreslagen metod för VCC tas fram. För att testa och illustrera den slutgiltiga meto-den genomförs en fallstudie på en produkt. Arbetet rundas av med diskussioner kring DFMA och den föreslagna metoden ur både hållbarhets-synpunkt och motivationssynpunkt. Samtliga resultat och diskussioner överlämnas sedan till företa-get för att möjliggöra vidare undersökningar kring en eventuell implementation av DFMA. / DFMA FMEA DFM DFA Lean Produktdesign Metodutveckling

MALDI imaging allows the creation of a molecular image of a tissue slice. This image is reconstructed from the ion abundances in spectra that are obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method that can provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. My hypothesis was that a method based on multiple reaction monitoring (MRM) with isotopically-labelled internal standards can be developed which would allow the accurate quantitation of proteins in MALDI Imaging. This study reports on the development of this novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labelled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabelled proteolytic peptides to the corresponding transitions from the applied isotopically-labelled standard peptides. In a parallel experiment, the quantity of the labelled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generated by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based imaging show the feasibility of obtaining precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins. / Graduate

Fatty acid profiling provides information on dietary intakes and an understanding of lipid metabolism. High throughput techniques such as fingertip prick (FTP) sampling has gained popularity in recent years as a simplified method for basic research, and could be further used to assess disease risk in the population, and other similar high-throughput techniques have the potential to assist in the monitoring and labeling of fatty acids in the food supply. With the advancement of high-throughput sample analysis techniques, a more complete understanding of storage stability is required as a larger volume of samples are produced with equal amounts of time to analyze them. Energy-assisted analysis techniques have the potential to help ameliorate some of these issues. Presently, FTP blood, whole blood and salmon storage stability is assessed under various storage conditions, and both microwave-assisted direct transesterification and indirect ultrasound-assisted extraction techniques are assessed. It is determined that storage of FTP blood and whole blood samples at -20°C results in significant and nearly complete highly unsaturated fatty acid (HUFA) degradation compared to all other temperatures examined. This degradation is determined to be the result of hemolysis and subsequent iron release from erythrocytes initiating fatty acid peroxidation reactions. Direct transesterification of FTP blood is reduced from as long as three hours to one minute with microwave-assisted energy and fatty acid extraction from ground flaxseed is reduced to 40 minutes from as long as 24 hours without compromising fatty acid profiles. Results of the current study provides insight into the storage stability of food sample and blood samples collected via high-throughput techniques, and provides support for the utilization of further high-throughput energy-assisted analytical methods that can help to minimize the potentially detrimental effects that long-term storage can have on fatty acid profiles.

<p>Mass spectrometry-based techniques have been widely used in reaction monitoring due to their high sensitivity and ability to offer structure information by tandem mass spectrometry. We applied nanoelectrospray mass spectrometry (nanoESI-MS) to simultaneously monitor pre-catalysts, catalytic intermediates, reagents, and products of palladium catalyzed Suzuki-Miyaura cross-coupling reactions. A set of Pd cluster ions related to the monoligated Pd (0) active catalyst is detected, and its deconvoluted isotopic distribution reveals contributions from two neutral molecules. One is assigned to the generally accepted Pd (0) active catalyst, seen in MS as the protonated molecule, while the other is suggested to correspond to a deactivated form of Pd catalyst. Oxidative stress testing of the synthetic model catalyst XPhos Pd cyclo-octadiene, performed using oxygen and Fe(III), supported this assignment. Thus, the make-up of the monoligated set of Pd (0) ions appears to indicate the oxidation state of the system. The formation and removal of the oxidative addition intermediate during the catalytic cycle was monitored to provide information on the progress of the transmetalation step. </p> <p> </p> <p><a>Recently, microdroplets created by ambient ionization source have been used as reaction vessels to accelerate organic reactions. Field desorption mass spectrometry under ambient conditions is applied to study solution-phase organic reactions in micro-volumes. Compared to nanoelectrospray, it is noteworthy that radical cations and formation of radical cation products are observed. Three reactions, the hydrazone formation by phenyl hydrazine and indoline-2,3-dione, the Katritzky reaction between a pyrylium salt and anisidine, and the Hantzsch synthesis of 1,4-dihydropyridine, were investigated by this system and reaction acceleration was observed to different extents. The increase in rate relative to that for the corresponding bulk reactions is attributed to solvent evaporation which increases concentration, and to the increase of surface-to-volume ratio with enhanced interfacial reaction rate constants. Later work in this thesis describes explicit solvent calculations to study the energies and structures of the hydrazone formation reaction from phenylhydrazine and indoline-2,3-dione in acidic methanol with density functional tight binding (DFTB) methods. Additionally, the thesis covers MS based methods for determination of isoaspartate and aspartate in peptide by gas-phase chemistry and detection of S-nitrosoglutathione in exhaled breath condensate sample.</a></p>

L’analyse des extraits naturels est de nos jours réalisée à l’aide de nombreuses techniques d’analyse plus ou moins complexes. Parmi ces techniques, l’HPTLC est bien connue en tant qu’outil d’identification phytochimique. Le but de cette thèse a été d’étudier l’apport de cette technique dans l’analyse des extraits naturels en dehors de son aspect « identification ». Ce travail a conduit à différentes études permettant de mettre en évidence des applications peu connues de l’HPTLC comme par exemple son utilisation comme technique semi-preparative. De plus, sa capacité à avoir une phase stationnaire résistante à différente matrices a été testée. Il a ainsi pu être démontré que cette caractéristique lui donnait l’avantage de procéder à des préparations d’échantillon plus minimaliste que les autres techniques de chromatographie. L’aspect quantitatif a été évalué et comparé à l’HPLC et a ainsi pu démontrer que des résultats équivalents sont obtenus, néanmoins, le manque de résolution et de précision ne permet pas à l’HPTLC d’attendre les exigences parfois demandées. Enfin, l’analyse d’un grand nombre d’échantillon a permis de montrer un des avantages de l’HPTLC dans le retraitement de données, que ce soit par regroupement visuel des profils obtenus ou par retraitement statistique / The analysis of natural extracts is nowadays performed with numerous techniques more or less complex. Among these techniques, the HPTLC is well known as a tool for phytochemical identification. The aim of this thesis was to study the contribution of this technique in the analysis of natural extracts outside its "identification" appearance. This work led to various studies to highlight little known applications of HPTLC such as its use as a semi-preparative technique. In addition, its ability to have a robust stationary phase to different matrices was tested. It has been demonstrated that this characteristic gave the advantage of proceeding more minimalist sample preparation than other chromatographic techniques. The quantitative aspect was evaluated and compared with HPLC and was able to demonstrate that similar results are obtained, however, the lack of resolution and accuracy do not allow the HPTLC has to wait sometimes requested requirements. Finally, the analysis of a large number of samples has allowed to show an advantage of the HPTLC in the data reprocessing, whether by visual grouping or obtained by statistical retreatment profiles