Dissertations / Theses on the topic 'Synchrotron Radiation Infrared Microspectroscopy'

2009/2010
ABSTRACT DEVELOPMENT OF MICROFLUIDIC DEVICES FOR BIOMEDICAL APPLICATIONS OF SYNCHROTRON RADIATION INFRARED MICROSPECTROSCOPY by Birarda Giovanni The detection and measurement of biological processes in a complex living system is a discipline at the edge of Physics, Biology, and Engineering, with major scientific challenges, new technological applications and a great potential impact on dissection of phenomena occurring at tissue, cell, and sub cellular level. The present PhD Thesis dealt with the development of methodologies and technologies to transform InfraRed MicroSpectroscopy (IRMS) into a mature technique to observe in real time biological events, and improving its ability to perform in vitro bio-experiments under physiological conditions. This goal has been achieved through the exploitation of microfabrication techniques to realize lab-on-chip (LOCs) transparent both in the Infrared and Visible region (IR-Vis), which allows measuring living cells. Up to now, IRMS has been almost exclusively employed for studying fixed cellular samples or tissues, allowing acquiring only “still frames” of the phenomena under investigation. The reason for that is to be ascribed both to the spectroscopic difficulties in working in water based environment and to the manufacturing constrains of the most common IR transparent materials, that limit the design flexibility of LOC devices suitable for IR analysis. We have overcome the so called “water absorption barrier” by extending microfluidic concepts to calcium fluoride, implementing innovative fabrication solutions for the realization of custom devices for IRMS studies of living cells subjected to different chemical and physical stimuli. Exploiting the high brightness of Synchrotron Radiation (SR) IR sources, that allows sampling at diffraction limited spatial resolution, we demonstrated the feasibility of the detection of intra-cellular processes. In parallel, novel strategies for IR data acquisition and analysis have been developed, opening the possibility to execute novel original experiments. Our studies were focused on the immune system, and in particular in evaluating the biochemical rearrangements characterizing human circulating leukocytes during their deformation, either when induced by purely mechanical stimuli or in response to a chemical gradient. Thanks to the microfabrication approach, we were able to mimic the cellular microenvironment both for studying pressure-driven micro-capillary circulation and chemically-driven extravasations of white blood cells. The present Thesis demonstrates that the “synergy of micro-approaches”, or rather the combination of micro-fabrication and IR micro-spectroscopy, can be exploited for extending the frontiers of Fourier Transform Infrared Spectroscopy (FTIR) to unexplored fields of life sciences. Through the careful control of the cellular microenvironment, crucial for an accurate data analysis as well as fundamental for the reliability of biological conclusions, some light could be shed on phenomena never investigated with IRMS, such as mechano-biology we directly explored, pulling down the water-barrier.
RIASSUNTO SVILUPPO DI DISPOSITIVI MICROFLUIDICI PER APPLICAZIONI BIOMEDICHE DELLA MICROSPETTROSCOPIA INFRAROSSA CON RADIAZIONE DI SINCROTRONE di Birarda Giovanni L’identificazione e la quantificazione di processi biologici in un complesso sistema vivente può essere ritenuta una disciplina al confine tra la Fisica, la Biologia e l’Ingegneria, con importanti sfide scientifiche, innovazioni tecnologiche e un grande impatto sulla dissezione di fenomeni a livello tissutale, cellulare e sub cellulare. Il presente lavoro di Dottorato ha avuto come obiettivo lo sviluppo di metodologie e tecnologie atte a rendere la MicroSpettroscopia InfraRossa (MSIR) una tecnica matura allo studio in tempo reale di fenomeni biologici, permettendo di effettuare esperimenti “in vitro” in condizioni fisiologiche. Questo obiettivo è stato raggiunto tramite l’utilizzo delle tecniche di microfabbricazione per la realizzazione di un “Lab-on-Chip” (LOC) trasparente sia nella regione dell’infrarosso che nel visibile, tramite il quale misurare cellule vive. Infatti fin’ora la MISR è stata impiegata quasi esclusivamente per lo studio di campioni di tessuti o di cellule fissati, permettendo di registrare solo “singoli fotogrammi” dei fenomeni sotto indagine. La ragione di questa limitazione è da imputarsi alle difficoltà spettroscopiche che si incotrano nell’investigazione di sistemi acquosi e ai limiti di fabbricazione dei più comuni materiali IR trasparenti, che hanno limitato la flessibilità di design necessaria alla realizzazione di LOC adatti alle analisi tramite MSIR. Siamo riusciti a superare la cosiddetta “barriera di assorbimento dell’acqua” tramite l’estensione dei concetti della microfluidica e dellamicrofabbricazione al calcio fluoruro, implementando soluzioni fabbricative che hanno permesso lo studio tramite MSIR di cellule viventi sottoposte a differenti stimoli sia di natura chimica che fisica. Grazie all’alta brillanza della Radiazione di Sinctrotrone (SR) IR, che permette il campionamento con una risoluzione spaziale al limte di diffrazione, abbiamo dimostrato la fattibilità dell’individuazione dei processi intra celluari. Contemporaneamente sono state sviluppate nuove strategie per l’acquisizione dei dati e per la loro analisi, permettendo il design di esperimenti innovativi. I nostri studi si sono concentrati sullo studio del sistema immunitario, in particolare nella valutazione della risposta biochimica caraterristica dei leucociti circolanti durante la loro deformazione, sia indotta da cause di tipo puramente meccanico, sia in risposta a gradienti chimici. Grazie alla microfabbricazione, siamo stati capaci di simulare il microambiente cellulare sia per lo studio dei globuli bianchi durante la circolazione microcapillare sia durante l’extravasazione indotta da gradienti chimici. La presente Tesi dimostra che la sinergia dei “micro” approcci, o piuttosto la combinazione di microfabbricazione e microspettroscopia IR, può essere usata per estendere le frontiere della MSIR a nuovi campi nello studio delle scienze della vita. Attraverso il preciso controllo del microambiente cellulare, cruciale per un’accurata analisi dei dati e fondamentale per l’attendibilità delle conclusioni biologiche, si possono chiarire fenomeni finora mai investigati tramite MSIR, come la meccano-biologia che abbiamo esplorato direttamente, abbattendo la barriera dell’acqua.
XXIII Ciclo
1981

Carcinome hépatocellulaire (CHC) est le sixième cancer et la deuxième cause de mortalité par cancer dans le monde. Dans la majorité des cas, le CHC se développe sur une maladie chronique associée à des étiologies variées telles que l'infection par le virus de l'hépatite B ou l’hépatite C, la consommation excessive d'alcool et des maladies métaboliques. Le développement des maladies chroniques du foie qui conduisent à la cirrhose puis au cancer induisent des modifications de la composition chimique des cellules et des tissus. En effet, la carcinogenèse hépatique est un processus en plusieurs étapes caractérisé par la progression de nodules de régénération, de nodules dysplasiques de bas grade puis de grade et enfin du CHC. Le traitement du CHC reste difficile et la transplantation du foie est la seule option thérapeutique curative à long terme. Le problème est qu'il n'y a pas de marqueur objectifs et quantifiables pour contrôler la qualité d’un greffon. Des biomarqueurs spécifiques marquant la progression du CHC font également défauts.Dans ce travail de thèse, nous avons évalué l’intérêt de la microspectroscopie infrarouge (IR) pour le diagnostic de la stéatose, qui est le facteur le plus important affectant la reprise de la fonction hépatique après greffe de foie. La microspectroscopie infrarouge permet de détecter de façon qualitative et quantitative les caractéristiques biochimiques liées aux différents constituants moléculaires présents dans l'échantillon biologique. Nos travaux ont montré que la progression de la stéatose hépatique correspond non seulement à l'accumulation de lipides, mais également à des changements spectaculaires dans la composition qualitative du tissu. En effet, le bas grade de stéatose présente une diminution de la teneur en glycogène et une augmentation concomitante de lipides par rapport au foie normal. La stéatose intermédiaire montre une augmentation de glycogène et des changements majeurs sont observés en ce qui concerne les lipides, avec une contribution significative des acides gras estérifiés, des chaînes de carbone allongées et des lipides insaturés. Ces caractéristiques sont encore plus prononcées dans les hauts degrés de stéatose. De plus, nous avons mis en évidence que des changements biochimiques majeurs se produisent dans la partie non-stéatosique du tissu malgré son aspect normal sur le plan histologique, ce qui suggère que l’organe dans son ensemble reflète le degré de la stéatose.La deuxième partie de la thèse est focalisée la carcinogenèse hépatique. Il s’agit d’un processus en plusieurs étapes qui se caractérise dans la plupart des foies cirrhotiques par la progression de nodules hyperplasiques de régénération vers des lésions précancéreuses telles que les nodules dysplasiques de bas grade puis de haut grade et enfin le CHC. Le diagnostic différentiel entre nodules dysplasiques en particulier de haut garde et CHC reste extrêmement difficile. Nous avons abordé le potentiel de la microspectroscopie IR pour le diagnostic des nodules cirrhotiques. Nous avons observé de profondes modifications de la composition biochimique du foie pathologique. En effet, des changements importants ont été détectés dans la composition des lipides, des protéines et des sucres mettant en évidence la reprogrammation métabolique dans la carcinogenèse. Les principaux changements ont été observés dans le domaine de fréquence 950-1480 cm-1 dans lequel plusieurs bandes permettaient la discrimination des nodules cirrhotiques, dysplasiques et tumoraux. Enfin, nous avons montré que le diagnostic peut être réalisé à l’aide d’un microscope de laboratoire qui peut être facilement mis en œuvre en milieu hospitalier
Hepatocellular carcinoma (HCC) is the sixth most common neoplasm and the second most common cause of death in the world. Hepatocarcinogenesis is a multistep process characterized in patients with chronic liver diseases by a spectrum of hepatic nodules that mark the progression from regenerative nodules to dysplastic lesions followed by HCC. Liver transplantation remains the curative therapeutic option able to treat both the HCC and the underlying liver disease. The issue is that there is no objective and quantifiable marker for quality control of liver graft. Specific biomarkers of early stages of HCC are also an unmet need.In this study, we have evaluated the potential of infrared (IR) microspectroscopy for the diagnosis of steatosis, one of the most important factors affecting the liver allograft function. Vibrational microspectroscopy, such as Fourier transform infrared microspectroscopy (FTIR), allows detecting spectral characteristics associated with different molecular components present in the biological sample, both qualitatively and quantitatively. Our first working hypothesis was that the progression of liver steatosis corresponds not only to the accumulation of lipids but also to dramatic changes in the qualitative composition of tissue. Indeed, a lower grade of steatosis showed a decrease in glycogen content and concomitant increase in lipids in comparison with normal liver. Intermediate steatosis exhibited an increase in glycogen and major changes in lipids, with a significant contribution of esterified fatty acids with elongated carbon chains and unsaturated lipids, and these features were more pronounced in a high grade of steatosis. Furthermore, we have shown, that FTIR approach allows a systemic discrimination of morphological features, leading to a separate investigation of steatotic vesicles and the non-steatotic counterpart of the tissue. This highlighted the fact that dramatic biochemical changes occur in the non-steatotic part of the tissue also despite its normal histological aspect, suggesting that the whole tissue reflects the grade of steatosis. The second part of the thesis focused on hepatocarcinogenesis; a multistep process that is characterized in most cirrhotic livers by the progression from hyperplastic regenerative nodules to low grade dysplastic nodules (LGDN), high grade dysplastic nodules (HGDN) and finally small HCC which corresponds either to vaguely nodular well differentiated HCC so called early HCC or to distinctly nodular moderately differentiated hepatocellular carcinomas. Since the differential diagnosis between precancerous dysplastic nodules and early HCC remains extremely difficult, we addressed the potential of FTIR microspectroscopy for grading cirrhotic nodules. The study was focused on 39 surgical specimens including normal livers as controls, dysplastic nodules, early HCC and the progressed HCC. Profound alterations of the biochemical composition of the pathological liver were demonstrated by FTIR microspectroscopy. Indeed, dramatic changes were observed in lipids, proteins and sugars highlighting the metabolic reprogramming in carcinogenesis. The major changes were observed in the frequency domain 950-1480 cm-1 in which several bands allowed significant discrimination of cirrhotic nodules, dysplastic lesions and HCC. Finally, a significant discrimination between benign, dysplastic nodules and early HCC remained possible using a FTIR microscope equipped with a laboratory-based infrared source that can be easily implemented in hospital environment. In conclusion, our study positions FTIR microspectroscopy as a versatile and powerful approach for investigating liver diseases, such as steatosis, dysplastic lesions and cancer. Further studies on larger series of patients as well as on biopsies will allow confirming the clinical reliability of such spectral signatures. Therefore, we anticipate that FTIR microspectroscopy will open new avenue in clinical diagnosis

Lors de ce travail nous avons étudié l'effet photodynamique (PDT) de l'Hypocrelline A (HA) sur quatre lignées de cellules cancéreuses (HeLa, Calu-1, K562, K562 RSTI571) par une approche biophysique. Nous avons combiné les informations obtenues par microscopie de fluorescence confocale, tests de cytotoxicité et microspectroscopie infrarouge à transformée de Fourier (IRTF) pour éclairer la physico-chimie de la PDT. La très haute sensibilité de la microscopie de fluorescence confocale permet de détecter la fluorescence intrinsèque de HA à des concentrations de l'ordre du nanomolaire, de suivre sa pénétration dans les cellules et de réaliser des cartes 3D haute résolution montrant sa distribution. La microspectroscopie IRTF est utilisée à fin de détecter des changements de la composition chimique globale de la cellule et les modifications de la structure des principaux composants cellulaires lors du traitement. L'utilisation d'une source synchrotron autorise une résolution subcellulaire qui devrait permettre de discriminer les réactions à l'intérieur des différents compartiments cellulaires tel que le noyau et le cytoplasme. Les tests de cytoxicité et l'utilisation de marqueurs fluorescents ont permis de comparer les effets des traitements croissants sur les différentes lignées cellulaires et d'optimiser les conditions de traitement pour obtenir différents types de mort cellulaire. Nous avons également montré que la combinaison de la microspectroscopie IRTF et de la microscopie de fluorescence sur un même instrument était possible et permettait de mesurer simultanément le spectre IRTF d'une cellule unique et le type de mort cellulaire qu'elle a subi. Les différentes approches utilisées dans ce travail se combinent pour montrer que HA ne pénètre pas dans le noyau mais est localisée dans le cytoplasme et accumulée autour du noyau après 24 heures d'incubation ; que les 4 lignées cellulaires réagissent de façon similaire ; que des modifications de la composition chimique des noyaux des cellules sont détectables par microspectroscopie IRTF mais que les cytoplasmes des cellules adhérentes sont trop minces pour y détecter des altérations sauf dans la région périnucléaire. Même en absence de pénétration de HA dans le noyau, la microspectroscopie IRTF permet de détecter des changements de la structure secondaire des protéines du noyau et des perturbations des membranes du réticulum endoplasmique et de l'appareil de Golgi sous l'action de espèces oxygénées réactives générées par la PDT
In this thesis, we studied the photodynamic effects of Hypocrellin A on four cancer cell lines using fluorescence microscopy, cytotoxicity tests and FT-IR microspectroscopy. As HA is a natural fluorescent substance, we used this intrinsic property to observe its localization in HeLa cells by fluorescence microscopy. The fluorescence images revealed that HA did not penetrated into the nucleus but localized in the cytoplasm and aggregated perinuclearly after 24 hours of incubation. MTT viability assay was performed to evaluate the optimum PDT conditions (HA and light dose) which induced cell death in the four cell lines HeLa, Calu-1, K562, K562 RSTI571. The dark toxicity of HA on ail cell lines is low, but is increasing dose-dependently. The phototoxicity of HA on all cell lines was increasing in both HA dose and light dose dependent manner. The light irradiation alone affected only negligibly the cell survival. HeLa cells are sensitive to HA PDT than Calu-1 cells. We found also that K562 and K562 RSTI are sensitive to HA. Moreover the synergetic effect of HA and Glivec® on K562 RSTI was observed. FT-IR microspectroscopy detected the changes in the secondary structure of proteins exhibiting an increase of beta sheets characteristics frequency affected by ROS generated from PDT. , with a predominant shoulder at around 1630 cm"1 (for the Amide I band) and 1530 cm"1 (for the Amide II band). Moreover, a slight decrease of the lipid intensity was noticed. Coupling fluorescence microscopy and FT-IR microscopy was carried out on the same instrument. Fluorescence microscopy could reveal the modes of cell death while FT-IR microspectroscopy showed effect of HA PDT on the secondary structure of proteins. All approaches carried out in this thesis revealed that even HA did not penetrate in the nucleus but there are changes in secondary structure of proteins in nucleus which can be observed by FT-IR spectromicroscopy

Disease- and drug-related bone disorders are rapidly increasing in the population. The drugs which are used for the treatment of neurodegenerative diseases and metabolic derangements, may have negative or positive effects on bone tissues. In the first study, the possible side-effects of Carbamazepine and epileptic seizures on bone structure and composition were investigated by FTIR and synchrotron-FTIR microspectroscopy, AFM and micro- and nano-hardness analysis. The effects on the blood parameters, bone turnover and vitamin D metabolism were also investigated by ELISA and western blot analysis. The current study provides the first report on differentiation of the effects of both epileptic seizures and AED therapy on bones. Besides Carbamazepine treatment, seizures also caused a decrease in the strength of bone. The biochemical data showed that both the epileptic and drug-treated groups decreased vitamin D levels by increasing the vitamin D catabolism enzyme
25-hydroxyvitamin D-24-hydroxylase. In the second study, the possible pleiotropic (positive) effects of cholesterol lowering drug
Simvastatin on bones were investigated by ATR-FTIR spectroscopy. The current study provides the first report on dose-dependent effects of simvastatin on protein structure and lipid conformation of bones. ATR-FTIR studies showed that although both high and low dose simvastatin strengthen bones, low dose simvastatin treatment is much more effective in increasing bone strength. Neural network analysis revealed an increased antiparallel and aggregated beta sheet and random coil in the protein secondary structure of high dose group implying a protein denaturation. Moreover, high dose may induce lipid peroxidation which limit the pleiotropic effects of high dose treatment on bones. This study clearly demonstrated that using low dose simvastatin is safer and more effective for bone health than high dose simvastatin treatment.

Hfq est une protéine bactérienne qui a un rôle pleiotropique. La principale fonction de la protéine Hfq bactérienne consiste à répondre aux stress que peut rencontrer la bactérie lors d’un changement environnemental, en utilisant essentiellement un contrôle post-transcriptionnel. La protéine, par sa capacité à interagir avec les ARN et notamment les petits ARN non codant, permet ainsi une régulation rapide de l’expression génétique. En outre la protéine interagit aussi avec l’ADN qu’elle aide à se structurer. Les mutations dans le gène qui code pour Hfq ont des effets pleïotropes (déterminant plusieurs caractères phénotypiques).D’un point de vue structural, la protéine adopte un repliement de type Sm, caractérisé par un oligomère toroïdal reposant sur la formation d’un feuillet β continu à 30 brins. Cependant, outre cette région Sm N-terminale, Hfq possède également une région C-terminale (CTR) de taille et de séquence variables selon les bactéries. Mon travail de thèse a porté sur l’analyse de cette région CTR chez la bactérie Escherichia coli. Cette région a en effet la capacité de former une structure de type amyloïde : structures auto-assemblées in vivo, à proximité de la membrane interne et dans le nucléoïde.Par l’utilisation de diverses techniques physico-chimiques (microscopie moléculaire, spectroscopie et microscopie infrarouge, dichroïsme circulaire et diffusion aux petits angles), mon travail a consisté à caractériser l’assemblage de cette région de Hfq ainsi que les facteurs l’influençant en particulier la présence d’acide nucléique. Une partie de mon travail de thèse a aussi consisté à mettre en place une méthode d’imagerie corrélative innovante permettant d’analyser la signature chimique et morphologique d’une fibre amyloïde unique. Mon travail a enfin porté sur l’analyse de l’effet de composés inhibant l’agrégation de la structure amyloïde, ce qui pourrait constituer une piste pour développer une nouvelle classe d’antibiotiques
Hfq is a pleiotropic bacterial protein that determines several phenotypic characteristics. Its main function is to facilitate responses to stresses that bacteria may encounter during environmental changes, mainly by using post-transcriptional genetic control. The protein, by its capacity to interact with RNA, in particular small non-coding RNA, enables a rapid regulation of gene expression. In addition, the protein also interacts with DNA and compacts it. From a structural point of view, the protein adopts an Sm-like fold, characterized by a toroidal oligomer formed by a continuous 30-stranded β-sheet. Besides its conserved N-terminal Sm domain, Hfq also possesses a C-terminal region (CTR) that can vary in size and sequence between bacteria. My PhD work focused on the analysis of this CTR region in Escherichia coli bacteria. Indeed, this region has the capacity to form an amyloid structure. This structural dynamic is related to the formation of self-assembled structures in vivo, in the proximity of the inner membrane and in the nucleoid.Using various physicochemical techniques (molecular microscopy, spectroscopy and infrared microscopy, circular dichroism and small angle X-ray scattering), my work consisted in characterizing the assembly of this region of Hfq, as well as the factors influencing its assembly (in particular, the presence of nucleic acids). A part of my work consisted in setting up an innovative correlative–imaging method to analyze the chemical and morphological signature of a single amyloid fibre. Finally, my work focused on the analysis of the effect of compounds that inhibit the aggregation of the amyloid structure, which could constitute a new way to develop a novel class of antibiotics

La mesure des concentrations d’espèces à l’état de trace, susceptibles d’avoir un impact notable sur la santé, le climat ou la stabilité de la couche d’ozone constitue un véritable défi. Les prochaines missions spatiales, prévues à haute sensibilité, apporteront un progrès si et seulement si les paramètres spectraux nécessaires sont disponibles. Pour certaines espèces d’intérêt atmosphérique telles que le chlorure de nitryle (ClNO₂), le phosgène (Cl₂CO) et le nitrate de chlore (ClONO₂), les données spectroscopiques sont incomplètes ou quasiment inexistantes. Le défi dans cette thèse consiste à obtenir des paramètres spectroscopiques (positions et intensités de raies ou sections efficaces d’absorption) pour ces espèces, en vue des applications atmosphériques. Cependant, hormis Cl₂CO, la spectroscopie de ClNO₂₂ et ClONO₂ est rendue difficile par leur synthèse chimique très complexe, leur réactivité avec les métaux et les matériaux organiques et leur instabilité en présence de lumière et chaleur. De plus, ces molécules sont assez lourdes (présence du chlore avec ses deux isotopomères) et présentent des spectres denses et assez compliqués par de nombreuses perturbations qui affectent les niveaux de vibration-rotation.Dans le cas de ClNO₂, des spectres ont été enregistrés dans la gamme 300 – 900 cm-1 avec de meilleures conditions expérimentales (haute résolution, basse température, long parcours, faible pression) en utilisant le rayonnement synchrotron de la ligne AILES à SOLEIL. Des modélisations précises des régions à 370 et 790 cm-1 ont été effectuées. Ces régions pourront être exploitées pour une future télédétection atmosphérique par spectroscopie IRTF respectivement par les instruments FORUM et IASI-NG. Les vibrations de basse énergie de ClONO₂ jamais observées à haute résolution avant ce travail ont également été mesurées. Une première modélisation de la région de la torsion vers 120 cm-1 est présentée dans cette thèse. L’analyse de ces vibrations sera utile pour modéliser les bandes chaudes dans les fenêtres atmosphériques où ClONO₂ est actuellement détecté, et in fine permettra d’aboutir à une restitution bien plus précise du profil de concentration de ClONO₂. Concernant Cl₂CO, des sections efficaces ont été mesurées au LISA, d’une part, à température ambiante pour clarifier les données des travaux antérieurs et, d’autre part, dans les conditions stratosphériques en soutien à la télédétection satellitaire
Measuring concentrations of trace species that may have a significant impact on health, climate or the stability of the ozone layer, is a serious challenge. Future space missions, planned at high sensitivity, will bring progress if and only if the necessary spectral parameters are available. For some species of atmospheric interest such as nitryl chloride (ClNO₂), phosgene (Cl₂CO) and chlorine nitrate (ClONO₂), spectroscopic data are incomplete or almost non-existent. The challenge in this thesis is to get spectroscopic parameters (line positions and intensities or absorption cross sections) for these species in support of atmospheric applications. However, apart from Cl₂CO, spectroscopy of ClNO₂ and ClONO₂ is made difficult by their very complicated chemical synthesis, their reactivity with metals and organic materials, and their instability in the presence of light and heat. Moreover, these molecules are quite heavy (presence of chlorine with its two isotopomers) and exhibit dense spectra that are quite complicated by numerous perturbations affecting vibration-rotation levels.In the case of ClNO₂, spectra were recorded in the range 300 – 900 cm-1 with much improved experimental conditions (high resolution, low temperature, long path, low pressure) using the synchrotron radiation of the AILES beamline at SOLEIL. Precise modelling of the 370 and 790 cm-1 regions has been performed. These regions could be used for a future atmospheric remote sensing by FTIR spectroscopy respectively by FORUM and IASI-NG instruments. The low energy vibrations of ClONO₂ that have been never observed at high resolution before this work were also measured. A first modelling of the torsional region around 120 cm-1 is presented in this thesis. The analysis of these vibrations will be useful for the modelling of hot bands in the atmospheric windows where ClONO₂ is currently detected, and in fine will lead to a much more precise retrieval of the ClONO₂ concentration profile. Regarding Cl₂CO, cross sections have been measured at LISA, on one hand, at room temperature to compare data with previous works and, on the other, in stratospheric conditions to support satellite remote sensing applications

Biological tissues were studied with synchrotron infrared (IR) microspectroscopy, a technique that allows the spatially resolved determination and mapping of multiple components in situ at high spatial resolution. The first project involved studying brain tissue from TgCRND8 mice, a transgenic model of Alzheimer’s disease (AD). AD is the main cause of dementia in the ageing population, marked by the deposition of plaques composed of the Aβ peptide. Dense-cored and diffuse plaques were IR mapped and the results correlated with histochemistry and immunostaining. Spectral analysis confirmed that congophilic plaque cores were composed of highly aggregated protein in a β-sheet conformation. The amide I maximum of plaque cores was 1623 cm-1; there was no evidence of the high frequency (1680-1690 cm-1) peak seen in in vitro Aβ fibrils and attributed to anti-parallel β-sheet. A significant elevation in phospholipids was found around dense-cored plaques in TgCRND8 mice ranging in age from 5 to 21 months. This was due to an increase in cellular membranes from dystrophic neurites and glial cells around the core, but could also contribute to Aβ aggregation through the interaction of newly secreted Aβ with phospholipids. In contrast, diffuse plaques were not associated with infrared detectable changes in protein secondary structure or relative concentrations of other tissue components. In addition, focally elevated deposits of creatine, a molecule with a crucial role in energy metabolism, were discovered in AD brain tissue with IR microspectroscopy. The creatine deposits may be a previously undiscovered disease marker. A second project was part of a larger Natural Sciences and Engineering Research Council Collaborative Health Research Project (NSERC-CHRP) to test the hypothesis that treatment with anti-oxidants, L-2-oxo-thiazolidine-4-carboxylate (OTC) and quercetin, following spinal surgery may reduce oxidative stress, inflammation, and scarring. The effect of OTC and quercetin on scar tissue formation was evaluated in rats that had undergone laminectomy. Synchrotron IR microspectroscopy data were collected on scar tissue from OTC, quercetin and saline (control) treated animals, sacrificed at 3 and 21 days post-surgery. Spectral differences could be correlated with the stages of wound healing.
May 2007

The level of information provided by electrochemical measurements can be substantial as evident by the use of electrochemistry in varied disciplines spanning from materials research to cellular biochemistry. However, electrochemistry on its own does not provide direct information concerning redox induced changes in molecular structure. This information can only be elucidated by coupling spectroscopic and/or separation techniques with traditional electrochemical methodologies. In principle, infrared (IR) spectroelectrochemistry (SEC) is ideal for such studies but in practice coupling IR spectroscopy and electrochemistry are often experimentally incompatible. Since the inception of in-situ IR SEC techniques in the 1980’s, two competing methodologies (using either external- or internal- IR reflection geometries), were developed to deal with the two major challenges associated with IR SEC (strong infrared absorption of the electrolytes and weak analytical signals). The primary focus of this thesis is the successful advancement of IR SEC techniques through the implementation of synchrotron infrared radiation with ultramicroelectrodes (UMEs; electrode diameters < 25 µm) to study spectroelectrochemical processes on the microsecond time scale. Several examples using Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS) are presented including the adsorption of dimethylaminopyridine (DMAP) on gold substrates and the proton-coupled electron-transfer (PCET) kinetics of electrochemically-active 1,4-benzoquinone terminated self-assembled monolayers (SAMs). These studies highlight the benefits of coupling electrochemistry and infrared spectroscopy. For instance, in-situ spectroscopic evidence shows that small amounts of DMAP’s conjugate acid (DMAPH+) adsorb on gold electrodes in acidic electrolytes and at negative potentials. This result was not forthcoming from previous electrochemical measurements and was only realized through in-situ SEIRAS. Finally, the largest contribution in advancing in-situ IR SEC methodologies was through the development of utilizing synchrotron infrared radiation on UMEs to study fast electrochemical processes. This work was technically very challenging and emphasized the interfacing of an electrochemical cell containing an UME with fast infrared data acquisition techniques (i.e. rapid scan and step-scan interferometry). The use of a prototypical electrochemical system, i.e. the mass-transport controlled reduction of ferricyanide, indicate that at short times the spectroscopic signal closely matches the electrochemical signal but at long time scales it deviates due to edge effects associated with the diffusion environment of the UME.

碩士
國立交通大學
加速器光源科技與應用碩士學位學程
100
Fe3O4 and Fe3O4@SiO2 magnetic nanoparticles (MNPs) have recently become important in biomedical applications; however, influences of these MNPs to cells are still not very clear. Bare Fe3O4 and Fe3O4@SiO2 MNPs should be noticed because any surface modification may be removed from them when they enter into cells or in cells. In this work, in order to avoid too much surface residues from the precursors, coprecipitation method is adopted to synthesize bare Fe3O4 MNPs, while Stober process is performed to synthesize bare Fe3O4@SiO2 MNPs. The characterization of MNPs is indentified by X-ray Diffraction (XRD), Transmission Electron Microscopy (TEM), X-ray Photoelectron Spectroscopy (XPS), X-ray Absorption Spectroscopy (XAS) and Superconducting Quantum Interference Device Magnetometer (SQUID). These results show that as-prepared Fe3O4 MNPs primarily contains crystalline Fe3O4 phase, while the deposited SiO2 on Fe3O4 MNPs is amorphous. A549 lung cancer cells are used as model cells for MNPs treatment, and the cell viability is measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results show that mitochondrial reductase activity in cells is reduced by treating Fe3O4 MNPs and Fe3O4@SiO2 MNPs to A549 cells for 36 hr. Instead of traditional biochemical methods, synchrotron radiation infrared-ray (SRIR) spectra and synchrotron radiation infrared-ray microscopy (SRIRM) with high spatial resolution 10μm are carried out to measure the change of chemical components and chemical composition distribution in cells. These results exhibit that DNA structures in cells are indirectly affected by Fe3O4 MNPs and Fe3O4@SiO2 MNPs, and the concentration of DNA becomes less with MNPs concentration and treatment time while no protein and lipid changes are observed, but the lipid/protein ratio is MNPs-concentration-dependent and treatment-time-dependent and it is observed that the amount of lipids is relatively larger at far-nucleus regions while that of proteins is relative larger at and around the nucleus region.

The PhD thesis work has been focused on aprotic Li-O2 cells assembled with different cathode materials in combination with the ether-based LiTFSI/TEGDME electrolyte. The performance of these promising energy storage systems have been investigated and related to the electrochemical and chemical processes at the triple O2/cathode/electrolyte interface and to the peculiar influence of each type of cathode material. These goals have been achieved by a powerful combination of techniques such as X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and X-ray Photoelectron Spectroscopy (XPS). The remarkable role of the TFSI- anion in the degradation process of a carbon-based cathode has been discussed in Chapter 4 on the basis of a post-mortem characterization of the positive electrode. Moreover, the fade of long-term stability of the cells has been related to the accumulation on the cathode surface of both insulating reaction products and byproducts, leading to the rise of overvoltages upon discharge and charge. A full electrochemical and chemical characterization of novel carbon-free Mn+-doped (Mn+=Cr3+ and Zn2+) NiCo2O4 nanomaterials grown on Ni foam (NCCr@Ni and NCZn@Ni) has been reported in Chapters 5 and 6. For the first time in the literature these materials have been tested as cathodes in Li-O2 cells and the beneficial effect of Cr(III)- and Zn(II)-doping of the NiCo2O4 spinel to boost the kinetics of both the ORR and the OER has been demonstrated. By means of in-house and synchrotron measurements the dopants role in the increase of the performance respect to the undoped material has been investigated. The preparation and characterization of novel composite Pd/PdO@NCCr@Ni cathodes made of Pd nanoparticles deposited on a nanostructured Cr(III)doped NiCo2O4 on Ni foam has been described in Chapter 7. The synergistic co-catalytic effect between the Pd NP and the doped nickel cobaltite has been proved to reduce both discharge and charge overvoltages and to improve the cell calendar life. Overall, the general objectives of the PhD activity have been met, since three novel materials have been produced and demonstrated to be performing cathodes for Li-O2 cells. Moreover, this study allowed the acquisition of skills concerning laboratory practice and widely applicable electrochemical and chemical characterization techniques.

Tesis (Doctor en Física)--Universidad Nacional de Córdoba. Facultad de Matemática, Astronomía y Física, 2009.
Este trabajo ha tenido como propósito ofrecer resultados concluyentes y proponer nuevos puntos de referencia en el tema de la cristalización de fosfatos de calcio en un entorno biológico, específicamente, la formación de cálculo dental. Si bien ésta afección no es una enfermedad, de igual manera ha tomado la atención de científicos de diversas áreas, como la medicina, la biología y la química. A la pregunta de '¿por qué sería importante estudiar el fenómeno de cristalización en fosfatos biológicos?' habría tres respuestas. La primera, ayudaría a entender el fenómeno de formación de depósitos pétreos en la boca que si bien no implica gran molestia o dolor, es comprobado que es disparador de severas enfermedades bucales crónicas como la periodontitis, además, claves en el estudio de formación también podría ser aplicable a otros depósitos pétreos como cálculos renales que en la mayoría de los casos requiere intervención quirúrgica. La segunda, podría revelar aspectos fundamentales en la formación de tejido óseo que serían de utilidad en la terapia de rehabilitación o en la recalcificación de fracturas óseas y más aun la enfermedad frecuente de personas mayores, la osteoporosis. La tercera, entender la fisicoquímica del proceso ofrecería un gran aporte para la fabricación de mejores prótesis óseas y dentales con un muy bajo nivel de rechazo por el organismo.
José Amado Abraham.