Despite the scientific progress in the last decades in terms of therapeutic agents to fight cancer there is still the need of developing safer and more effective drugs. Developing an innovative drug is not only very expensive, but also highly time consuming; furthermore, the number of anticancer agents that fail in clinical trials with high attrition rates mainly caused by unexpected toxicity and lack of efficacy far outweighs those considered effective, which indicates that drug screening processes require further improvements. In this project the application of Fourier transform infrared microspectroscopy is evaluated in order to develop a spectral based model that could be used to describe the drug-cell interaction and also to discriminate between the metabolic modifications due to a particular drug and the inherent cell cycle of a cell. A computational method was built using the FTIR spectra from a highly resistant renal cell carcinoma cell line, Caki-2, in order to discriminate between the phases of the continuous cell cycle that cells undergo while proliferating in vitro. Such model enabled the discrimination of early events of the cell cycle (G0/G1 phase cells) from G2/M phase cells with a prediction accuracy of 90% and 92.9% respectively. On the other hand, when the RMieS-corrected FTIR spectra corresponding to G0/G1, S and G2/M phases were modelled, the algorithm was able to retrieve each stage of proliferation with 82.3%, 71.8% and 84.4% accuracy respectively. Although the average accuracy yielded by the method was relatively low compared with what has previously been reported in the literature, these results emphasize the need to correct the data from physical distortions due to size and prove the principle that it is possible to create a method for identifying different events of the cell cycle based on the data that the FTIR spectroscopy provides, as well as using the scattering profile characteristic of each phase of development. Once the underlying biochemistry of proliferating Caki-2 cells were characterised by FTIR, the cells were treated with 5-Fluorouracil and paclitaxel, two widely used cytotoxic agents known to induce cellular damage at S or G2/M phase of the cell cycle respectively. The FTIR spectra collected were analysed via multivariate and bivariate techniques. Results demonstrated that, after 24 hours of treatment at the IC50 concentration of each drug, Caki-2 cells displayed spectral features consistent with early stages of apoptosis. These spectral characteristics did not appear to be linked either to the drug’s mode of action or the cell’s cycle phase. The cell’s proliferation stage was not the main classification trend among the drug-treated spectra; nevertheless, the cell cycle phase of each drug-treated population was successfully retrieved by an optimized model capable to classify such phases with an average accuracy of 77.98%. Altogether, this study offers a new perspective when analysing FTIR data from single cells as a function of the cell cycle and also when investigating the biochemical response of a cell line to a given anticancer agent.

The purpose of this study was to investigate the feasibility of using Fourier transform mid-infrared (FT-IR) microspectroscopy, with attenuated total reflectance (ATR), to detect the presence of Fusarium spp., classify differences in ability to detect Fusarium spp. among the Canadian soil zones, determine whether Fusarium spp. can serve as an indicator for the concentration of deoxynivalenol (DON), and develop an equation that can predict DON within wheat kernels. Canadian Western Red Spring wheat kernels were pressed against an ATR crystal to obtain spectra. Five chemical functional groups were investigated. The ability of FT-IR-ATR microspectroscopy to detect the presence of Fusarium spp. was confirmed. The chemical composition, and the ability to identify Fusarium spp., differed in kernels grown among the soil zones. Several methods to use Fusarium spp. as an indicator for the concentration of DON content within the wheat kernels were attempted and found to be unsuccessful.

The purpose of this study was to investigate the feasibility of using Fourier transform mid-infrared (FT-IR) microspectroscopy, with attenuated total reflectance (ATR), to detect the presence of Fusarium spp., classify differences in ability to detect Fusarium spp. among the Canadian soil zones, determine whether Fusarium spp. can serve as an indicator for the concentration of deoxynivalenol (DON), and develop an equation that can predict DON within wheat kernels. Canadian Western Red Spring wheat kernels were pressed against an ATR crystal to obtain spectra. Five chemical functional groups were investigated. The ability of FT-IR-ATR microspectroscopy to detect the presence of Fusarium spp. was confirmed. The chemical composition, and the ability to identify Fusarium spp., differed in kernels grown among the soil zones. Several methods to use Fusarium spp. as an indicator for the concentration of DON content within the wheat kernels were attempted and found to be unsuccessful.

Vibrational spectroscopy, mainly infrared spectroscopy, has been applied to bacteria, yeast and archaea cells for many years, for example, for the purpose of developing a rapid method of identification. More recently microcolonies have been used for consistency with the preparation and culture protocols of traditional microbiological methods. Heterogeneity of microcolonies has not been well studied. Investigation of heterogeneity may provide detailed biochemical information leading to an understanding of how colonies grow and the link to the growth cycle. Investigation of regions within bacterial colonies using FT-IR microspectroscopy was applied to two prokaryotes, the Gram-negative archaeon Halobacterium salinarium and the Gram-positive bacterium Bacillus stearothermophilus. Two-dimensional maps of the entire colony and point maps, spectra taken from key regions such as the periphery and centre of the colony, were acquired. The approximate size of the colonies ranged from 250-950 μm. The infrared data for the Gram-negative archaeon, H. salinarium supports that for the Gram-positive bacterium, B. stearothermophilus, despite the microorganisms being of different domains and Gram types. It was concluded that the periphery of the microcolony approximately equates to the exponential growth phase (and possibly the lag phase) where the younger cells reside, while the centre approximates to the death and stationary phases. However the spatial resolution proved to be a limiting factor, so Raman microspectroscopy was employed to address this. Raman spectra across the diameter of microcolonies ranging from 100-300 μm of the pigmented bacterial species Halobacterium salinarium revealed variations in the carotenoid bands. It was suggested that these variations correspond to growth rings, which relate to the growth cycle and the consolidation and migration phase of the cells. The carotenoid variation was rather clear mainly due to the enhanced spectral intensity due to resonance with the laser excitation source. Hence, pigmented bacterial colonies are ideal to study by Raman spectroscopy. The results of this particular aspect of the research are to be published in the Journal of Raman Spectroscopy [1]. In addition, a connection between the consolidation and migration phases and the phases of the growth cycle has been postulated as a novel hypothesis to link the periodic dynamics of the colony and the growth mechanisms at the cellular level. The Raman microspectroscopic study was extended to non-pigmented bacterial colonies directly on the growth medium. This was a more difficult endeavour as the spectra taken do not have the resonance enhanced advantage of a pigmented bacterium. In addition the sampling volume can consist of variable amounts of growth medium thus decreasing the signal-to-noise ratio and reducing the accuracy of subsequent spectral calculations. However, this was overcome to a large extent by the use of confocal microscopy. The non-pigmented bacterial colonies investigated were Bacillus stearothermophilus and Bacillus subtilis. Analysis, by band area ratios and by chemometric approaches, of radial line map spectra of both Bacillus species revealed variation of nucleic acid concentration. The higher nucleic acid concentration is likely to be a result of cells in the exponential growth phase as rapid growth of new cells is occurring.

The ability to measure glucose transcutaneously and noninvasively is an exciting prospect. Such a procedure will offer a painless way of glucose self-monitoring improving the lives of people with diabetes by lowering the barriers to optimal glycemic control. The noninvasive measurements involve collecting near-infrared spectra (4000–5000 cm-1; 2.0–2.5 µm) of skin with two optical fibers in a transmission geometry. Previous results indicate that repositioning of the fiber optic interface adversely affects both precision and accuracy of such measurements. Slight movements of the interface increase prediction errors more than 2.5–fold when performed with a stationary rat model. In this dissertation, the chemical heterogeneity of skin tissue is explored as a possible cause for the sensitivity of the measurement to the position of the optical interface. Rat and human skin tissues are mapped by using combination near infrared spectra the to provide distributions of the major components of skin: water, collagen type I protein, fat, keratin protein, and two scattering terms of constant and slope. On the basis of the measured heterogeneity, sets of rat and human skin spectra are simulated to investigate the impact of repositioning the fiber-optic interface. Glucose predictions are analyzed for each location of the interface for a series of partial least squares (PLS) calibration vectors established for different locations on the skin. Significant increases in the measurement errors are observed for the situation where the PLS calibration models are generated from spectra associated with one location of the interface while subsequent measurements are performed at slightly locations of the skin matrix. These increases in prediction errors match the 2.5–fold increase observed in vivo. The impact of replacing the spectrum of bovine fat with spectra of native fat for both rat and human skin samples is established. Principal component analysis (PCA) of the spectral residuals reveals that the magnitude of the spectral residuals and the effects of tissue fat content on the quality of the linear regression were decreased. The key implication of the research detailed in this dissertation is that chemical heterogeneity of skin tissue must be considered in multivariate models intended for noninvasive glucose measurements.

Ce travail a été entrepris dans le but de déterminer les chaînes opératoires des matières colorantes néolithiques et de les intégrer dans une dynamique chrono-culturelle, à partir de deux sites-clés de l’arc liguro-provençal : Castellar – Pendimoun (5750-4900 cal. BCE) et Nice – Giribaldi (4550-4050 cal. BCE).L’étude technoéconomique montre des situations contrastées. Pendimoun voit, tout au long du VIe millénaire BCE, l’exploitation de trois roches locales aux caractéristiques physico-chimiques variées, traitées selon des schémas simples similaires pour fournir des produits adaptés à tous les besoins : hématite ou goethite, en quantité pour un usage technique ou de granulométrie fine pour exploiter les propriétés colorantes. Giribaldi est le siège d’une gestion dichotomique, où des matières colorantes locales préparées comme à Pendimoun, côtoient des matières exogènes, traitées à travers des actions spécifiques adaptées à leur cohérence.Enfin, de nombreux aspects symboliques ont été mis en évidence et caractérisés. Un bloc calcaire est sculpté et peint pour représenter un visage, selon une tradition peut-être héritée de pratiques proche-orientales. La teinte rouge est associée à la sphère funéraire. La coloration de productions céramiques techniquement très investies et vraisemblablement dédiées à des pratiques sociales particulières est aussi observée ; cette coloration est appliquée par incrustation de poudre durant les VBQ à Pendimoun et par peintures rouges ou noires et par incrustations blanches au Chasséen ancien à Giribaldi. / The aim of this research was to determine the “chaînes opératoires” of colouring materials in the north-western Mediterranean region during the Early and Middle Neolithic and to integrate them in chrono-cultural framework. Two major archaeological sites were selected: Castellar – Pendimoun rock-shelter (5750-4900 cal. BCE) and Nice – Giribaldi, an open-air site (4550-4050 cal. BCE).The techno-economic analysis reveals contrasting situations. At Pendimoun, three local colouring materials, presenting various physic-chemical properties, were used during the VI millennium BCE. They were processed identically according to simple schemes in order to provide a wide range of products: goethite or hematite, in quantities for utilitarian needs or in fine powder for colouring purposes.Giribaldi witnessed a dichotomous management. Some local geomaterials were crafted such as the ones in Pendimoun but exogenous rocks were also specifically processed depending on their cohesion. Furthermore, this overview is not diachronically uniform as bauxites progressively replaced other raw materials in Early Chassey stages.Last, several symbolical aspects have been highlighted and characterized. A calcareous block was carved and painted in red- and yellowed to portray an anthropomorphic “mask”. The colour red is associated with funeral activities. Starting at the SMP phases, the main use of color is aimed at the coloring of ceramics, the latter probably dedicated to specific social activities as suggested by the technical investment involved: red powder incrustation in ceramic (Pendimoun) and then by white powder incrustation in engraved decor, black and red painting (Giribaldi).

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Předložená dizertační práce se zabývá kombinací optických mikromanipulací s mikrospektroskopickými metodami. Využili jsme laserovou pinzetu pro transport a třídění živých mikroorganismů, například jednobuněčných řas, či kvasinek. Ramanovskou spektroskopií jsme analyzovali chemické složení jednotlivých buněk a tyto informace jsme využili k automatické selekci buněk s vybranými vlastnostmi. Zkombinovali jsme pulsní amplitudově modulovanou fluorescenční mikrospektroskopii, optické mikromanipulace a jiné techniky ke zmapování stresové odpovědi opticky zachycených buněk při různých časech působení, vlnových délkách a intenzitách chytacího laseru. Vyrobili jsme různé typy mikrofluidních čipů a zkonstruovali jsme Ramanovu pinzetu pro třídění mikro-objektů, především živých buněk, v mikrofluidním prostředí.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / Isogenic waxy wheat lines differ from their non waxy (normal) parents in functionality, end use, and chemical (i.e. amylopectin/amylose, lipid) contents. Other investigations of waxy and parent wheats involved the carbohydrate and protein fractions. The goal of this work is to apply chemical images to discriminate between the waxy and parent wheats and define the contribution of contrasting lipid profiles. Recent waxy topics include current interest in plant breeding activity to develop new lines that incorporate desirable traits with advantageous success in baking and milling, and the differences needed in milling techniques for waxy versus normal wheats that may be associated with lipids. From our empirical preliminary success in sorting parent wheat kernels from waxy wheat full null specimens by nearIR chemical imaging it was anticipated that using fundamental vibrational spectra in the mid infrared would provide the chemical basis of discrimination. FT-IR microspectroscopic in situ probing and imaging of kernel frozen sections was applied to genetically pure, well documented isogenic breeding lines. With the use of high spatial resolution, elucidation of fundamental vibrations of mid IR provides chemical manifestation of the genetic expression that differentiates waxy wheats from their parent wheats. Comparison between numerous contiguous pixels, typically 3,000 for each type, establishes a consensus and a mean spectrum with characteristic bands for waxy and parent. Extractions with solvents of differing polarity were employed to aid in lipid extraction in situ and kernel extracted endosperm. Differences between kernel sections of waxy and parent are observed using FT-IR microspectroscopic imaging. However, revealing lipid class contribution to the molecular bands required infrared analysis after selective extraction. Triple mass spectrometry of lipid molecule ions was used for compositional analyses to enhance lipid class profile distinction. A normal and waxy advanced breeding line wheat were also analyzed via the same methods. It was noted that digalactoslydyglyercides are the most abundant lipids in all samples, however the relative lipid profiles of normal wheat versus waxy wheat differ as well as tetraploid versus hexaploid. It is observed that in the endosperm of all parent wheat versus waxy wheat specimens analyzed, all waxy wheat specimens contained higher lipid content. Methods were also applied to partial waxy isogenic cultivars to determine detection limits that correspond to the degree of waxy genetic expression.