The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants.

The above-ground organs of plants are covered by a protective cuticle, an extracellular membrane performing important physiological and ecological functions, that consists of cuticular wax and the fatty acid-derived polymer cutin. Until the past decade, the cuticular wax was thought to be a homogenous mixture. Hence, previous interpretations relating the chemical composition and biological functions of the cuticular wax were based on the total wax composition, an average taken over the entire area and depth of the cuticle. However, recent selective sampling experiments showed a heterogeneity of the chemical composition between different wax layers. The finding of this heterogeneity imposes the need for a more accurate description of the cuticle in order to understand how the chemical composition determines the biological function. This thesis is aimed at mapping the lateral patterns of cuticular waxes on Prunus laurocerasus leaf surfaces with microscopic resolution to provide spatially resolved chemical information in the interest of describing the cuticle more accurately. Firstly, this thesis examines the post-acquisitional data processing and analysis techniques followed by the investigation of the potential of Raman microspectroscopy for the simultaneous detection of structurally similar triterpenoids in plant cuticles. Relative composition analysis was performed on artificial triterpenoid mixtures and the resulting calculated triterpenoid ratios were consistent with the expected values. Qualitative and quantitative analysis of the linear near infrared (NIR) Raman, coherent anti-Stokes Raman scattering (CARS), and third harmonic generation (THG) spectra of isolated adaxial and abaxial P. laurocerasus cuticles demonstrated the in situ detectability of the triterpenoids using this approach. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentration above the periclinals and the guard cells. The main research contributions include evaluating various data processing techniques as candidates for automated implementation and applying different imaging techniques to obtain chemical information about the lateral concentration gradient of the triterpenoid components in the cuticles, with high spatial resolution. This thesis also provides the first direct (i.e. in situ) evidence for lateral spatial heterogeneity of triterpenoids in the cuticle of a model species, P. laurocerasus. This work is expected to impel further structure-function investigation of the cuticular membranes of plants. / Science, Faculty of / Chemistry, Department of / Graduate

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

Alzheimer Disease (AD) is a progressive neurodegenerative disorder characterized by memory loss and dementia. Both energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain. With synchrotron FTIR microscopy, extensive deposits of crystalline creatine (Cr) had been discovered in TgCRND8 mouse brain tissue by previous students in our lab. In this thesis, regions of hippocampus and caudate of 5 pairs of transgenic mice and their non-transgenic littermate controls were mapped using Raman and IR microspectroscopy to find clues to Cr origin in transgenic mouse brain. Raman spectra obtained at higher spatial resolution (1-2 µm) were used for better delineation of the Cr crystalline deposits and their environs. These results indicate that Cr crystals were formed after snap-freezing and desiccation of brain tissue. Therefore, it can be speculated that Cr might be exist in solution form in vivo.

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).

Bone marrow oedema is a term used to define the appearance of regions of low signal on T1 weighted and high signal on T2 weighted fat-suppressed magnetic resonance images. The potential association between bone marrow oedema and prognosis in pathologies such as osteoarthritis is becoming increasingly recognised through clinical studies. A limited number of clinical studies have linked bone marrow oedema to altered bone density or altered bone marrow perfusion. The principal aims of this study were to investigate these findings in vitro, using the equine forelimb. The presence of bone marrow oedema within the equine forelimb was initially confirmed by undertaking magnetic resonance imaging scans. Bone samples were selected from 10 animals, 5 exhibiting the presence of bone marrow oedema-type abnormalities (BMOA) at the distal metacarpal. Raman microspectroscopy was used to determine the chemical composition of bone and projection radiography to provide a measure of bone density. Micro computed x-ray tomography was undertaken on a subset of three bone samples exhibiting BMOA. A second component of the study utilised contrast enhanced magnetic resonance imaging to enable comparison of perfusion to bone marrow with and without evidence of oedema. A saline flushing agent containing Evan’s blue was used so that subsequent sectioning of the bone would enable visualisation of the distribution of contrast agent as part of a histological examination of the oedematous region. An initial observation was that the majority of bone marrow oedema that was observed in the distal metacarpal appeared in a consistent location, namely the postero-inferior aspect of the bone, corresponding to the point of greatest load thereby suggesting a potential relationship to forces upon the joint. The principal observations were that there appears to be increased bone volume densities in those bone samples with evidence of bone marrow oedema. The Raman microspectroscopy did not demonstrate any statistically significant differences in the chemical composition of bone. Hence the overall impression is that bone marrow oedema is associated with a greater volume of bone, although of similar maturity and composition. There was limited evidence of increased perfusion (suggestive of increased vascularity and / or hyperpermeability) in those samples with bone marrow oedema. This work suggests that these particular bone marrow oedema lesions are associated with bone changes and potentially vascular changes although the aetiology is currently unclear. Further work is needed to determine the clinical significance and prognosis associated with these particular lesions, and whether these findings can be replicated for bone marrow oedema demonstrated at other anatomical locations.

The incidences of obesity and type 2 diabetes and their complications are increasing globally. The presence of elevated circulating free fatty acids has been associated with the initial dysfunction of pancreatic beta cells and microvascular endothelial cells followed later by their demise. The aim of this thesis was to investigate the mechanisms by which demise occurs, and how it may be prevented. Palmitate, a saturated fatty acid, caused cell death in both INS-1 beta cells and HCMec/D3 microvascular cells, whereas the unsaturated fatty acid oleic acid did not cause cell death, and also protected against palmitate-induced toxicity. Etomoxir, the mitochondrial CPT1 inhibitor did not rescue INS-1 or HCMec/D3 cells from palmitate-induced toxicity suggesting that palmitate-induced toxicity does not occur via entry into the mitochondria. Cells were exposed to 2-bromopalmitate, a non-metabolisable fatty acid used to reduce the pool of cytoplasmic CoA, to determine whether palmitate-induced toxicity might be mediated by its ability to be activated. Pre-incubation with 2-bromopalmitate in INS-1 cells significantly prevented palmitate-induced cell death. These data suggest that the activation of palmitate with CoA might mediate cell death. Cell cycle analysis found that neither oleic acid nor palmitate caused an increase or decrease in cell proliferation in both INS-1 and HCMec/D3 cells. The data suggest that the mechanism of oleic acid-induced cytoprotection might not be via a pro-proliferative mechanism. INS-1 cells were imaged using spontaneous Raman microspectroscopy after 24-hour exposure to esterified and non-esterified fatty acids. Uni- and multi-variate analysis and spectral decomposition were carried out using a methodology optimised and validated which is presented in this thesis. The aim was to quantify changes, if any, in lipid disposition: distribution, intensity (as a measure of concentration) and composition after exogenous exposure to these fatty acids. Exposure to 0.125 mM palmitate showed a significant decrease in the percentage of lipid within the cells and a corresponding increase in the intensity of this lipid. This suggests that palmitate, alone, might be shuttled into lipid droplets. This was not observed when the cells were exposed to oleic acid, whereby an increase in the intensity of lipid was observed even though no significant change was observed in the percentage of lipid within the cells. When palmitate and oleic acid were combined, the composition of the lipid droplets changed such that the levels of palmitate decreased and the levels of oleic acid increased. These data suggest that oleic acid does not shuttle palmitate into lipid droplets. These data do not support the hypothesis that oleic acid protects against palmitate-induced cytotoxicity by shuttling palmitate into lipid droplets. The methyl esters of palmitate and oleic acid were employed to determine whether they would affect lipid disposition. No change in lipid distribution or intensity was observed when the cells were exposed to these fatty acids, validating the requirement for the free carboxyl oxygen for the covalent binding to glycerol for the formation of lipid droplets. These data also suggest that INS-1 cells cannot de-esterify esterified fatty acids.

Miniaturization of devices to study chemical interactions and processes in liquid samples has led to the emergence of microfluidics and construction of lab-on-a-chip systems. Present work was devoted to implementation, development and testing of microfluidic systems with detection by confocal Raman microscopy and surface enhanced Raman scattering under the conditions of training department. Several options of performing standard macroscopic measurements in microscopic scales were explored. A method for measuring thermal stability of biopolymers in microsystems with contactless detection of temperature has been designed and tested. Furthermore, possibilites for studying the SERS effect within microfluidic channels were explored. It was demonstrated that the microfluidic chips provide promising opportunity to study hydrodynamics of liquids at microscopic level and chemical reactions and kinetics.

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.