Effects of the hydration state on the mid-infrared spectra of urea and creatinine in relation to urine analyses

When analysing solutes by FTIR spectroscopy in attenuated total reflection (ATR) mode, drying of samples onto the ATR crystal surface can greatly increase solute band intensities and, therefore, aid detection of minor components. However, analysis of such spectra is complicated by the existence of alternative partial hydration states of some substances that can significantly alter their infrared signatures. This is illustrated here with urea, which is a dominant component of urine. The effects of hydration state on its infrared spectrum were investigated both by incubation in atmospheres of fixed relative humidities and by recording serial spectra during the drying process. Significant changes of absorption band positions and shapes were observed. Decomposition of the CN antisymmetric stretching (νas) band in all states was possible with four components whose relative intensities varied with hydration state. These correspond to the solution (1468 cm-1) and dry (1464 cm-1) states and two intermediate (1454 cm-1 and 1443 cm-1) forms that arise from specific urea-water and/or urea-urea interactions. Such intermediate forms of other compounds can also be formed, as demonstrated here with creatinine. Recognition of these states and their accommodation in analyses of materials such as dried urine allows more precise decomposition of spectra so that weaker bands of diagnostic interest can be more accurately defined

Differential sepectral signature of human saliva using ATR-FTIR epsctroscopy in exclusive breastfeeding and supporting breasfeeding infants

Saliva can inform the physiological and pathological state of the body. The systemic benefits of exclusive breastfeeding in newborns are well documented, however the effect of supporting brestfeeding comparing to exclusive brestfeeding on salivary components remain unclear. Altogether, the aim of the present study was compare the salivary composition in supporting breastfed and exclusive breastfed infants using attenuated total reflectance Fourier transform infrared (ATR-FTIR). 28 mothers consented to participation of their 0-4-month old infants. 18 infants were exclusive breastfed and 10 were partially breastfed (breastfed plus formula-fed). Saliva were collected through the negative pressure apparatus e two microlitres were analyzed by ATR-FTIR. Bands 1650 cm-1 and 1153 cm-1 which identified the υNH (Amide I) and C-O-C stretching (Carbohydrate groups) of supporting brestfeeding were lower (p0.05) in both groups, this analysis indicates reduction of amide I and glycoproteins (carbohydrate groups covalently attached to many different proteins) are reduced in SB infants which can be affect salivary functions.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação (Mestrado)A saliva pode informar estados fisiológicos e patológicos do corpo e isto permite sua aplicação para ações clínicas de uma odontologia personalizada. Os benefícios do aleitamento materno para redução da morbidade, redução de doenças infecciosas e prevenção de baixo peso em recém-nascidos estão bem caracterizados. A espectroscopia de infravermelho com transformada de Fourier acoplada a um sistema de reflectância total atenuada (ATR-FTIR) é um excelente método para análises biológicas, no entanto, aplicações em determinação do estado fisiológico estão apenas começando a emergir. Ainda não foram descritas alterações na composição salivar de crianças sob amamentação exclusiva em comparação com aleitamento parcial. Desta forma, no presente trabalho foi utilizada a espectroscopia ATR-FTIR para avaliar a saliva de bebês com até quatro meses de idade sob aleitamento materno exclusivo ou sob aleitamento misto (com associação de amamentação e fórmula) buscando identificar possíveis mudanças nos componentes salivares relacionados à alimentação. Os modos vibracionais 1650 cm-1 e 1153 cm-1, que identificam o alongamento com υNH (Amida I) e C-O-C (região de carbohidratos livres e conjugados), foram menores (p 0,05) em ambos os grupos, este trabalho demonstrou que a inserção de aleitamento misto promoveu redução da amida I e de glicoproteínas (grupos de carboidratos ligados covalentemente a diferentes proteínas) em comparação com aleitamento materno exclusivo. Este achado pode ser um importante indicativo para novas análises de proteômica e metabolômica que poderão se relacionar com alterações nas funções salivares de bebês sob diferentes formas de aleitamento

Utilisation de la spectro-imagerie IR-TF pour le développement d'une anatomo-pathologie moléculaire des tumeurs cérébrales

Les gliomes sont des tumeurs agressives de mauvais pronostic, très angiogéniques et infiltrantes ce qui rend leur exérèse particulièrement difficile. Vu les limites des techniques actuelles d imagerie, nous avons proposé la spectro-imagerie Infrarouge à Transformée de Fourier (IRTF), d une résolution spatiale de 6 m, pour apporter une information moléculaire à l examen histologique actuel des gliomes. Nos travaux ont été fondés sur la recherche de paramètres moléculaires des vaisseaux sanguins, notamment sur la base des contenus de leur membrane basale. Celle-ci subit des altérations dûes au stress angiogénique tumoral. Nous avons mis en évidence des altérations de la structure secondaire des protéines (tels les collagènes) des vaisseaux sanguins au cours de la croissance de la tumeur. Nous avons aussi évalué les modifications des chaines d acides gras des phospholipides membranaires, qui révélent un degré d insaturation plus important pour les vaisseaux tumoraux. Ensuite, sur un modèle de gliome murin, nous avons établi une méthode efficace de classification des capillaires sanguins sur la base d absorptions de leurs contenus glucidiques et lipidiques, permettant de discriminer totalement les capillaires sains et tumoraux. La combinaison de ces paramètres a été mise à profit pour assurer une histopathologie moléculaire des gliomes humains. Nos résultats ont démontré qu il est possible de différencier entre la vasculature saine et tumorale sur ces gliomes humains, ce qui permet une bonne délimitation des zones tissulaires correspondantes. Cette technique pourrait devenir un outil analytique fiable, rapide d une durée compatible avec la chirurgie et donc très utile pour les neurochirurgiens.Malignant gliomas are very aggressive tumors with poor prognosis, highly angiogenic and invasive into the surrounding brain parenchyma, making their resection very difficult. Regarding the limits of current imaging techniques, we have proposed Fourier Transform Infrared (FTIR) spectro-imaging, with a spatial resolution of 6 m, to provide molecular information for the histological examination of gliomas. Our work was based on the research of molecular parameters of blood vessels, notably on the basis of the contents of their basement membrane, which undergoes changes due to tumor angiogenic stress. We have identified alterations of the secondary structure of proteins (such as collagen) in blood vessels during tumor growth. We have also assessed the changes in fatty acyl chains of membrane phospholipids, which revealed a higher unsaturation level in tumor vessels. Then, on a murine glioma model, we have established an efficient method of blood vessels classification based on their carbohydrates and fats contents, allowing the differentiation between healthy and tumor blood vessels. The combination of these parameters was used to provide a molecular histopathology for the study of human gliomas. Our results have demonstrated the feasibility of differentiating between healthy and tumor vasculature in these human gliomas, which help delimitating areas of corresponding tissue. This technique could become a reliable and fast analytical tool, with duration compatible with the surgery and thus very useful for neurosurgeons.BORDEAUX1-Bib.electronique (335229901) / SudocSudocFranceF

Ultra-Filtration of Human Serum for Improved Quantitative Analysis of Low Molecular Weight Biomarkers using ATR-IR Spectroscopy

Infrared spectroscopy is a reliable, rapid and cost effective characterisation technique, delivering a molecular finger print of the sample. It is expected that its sensitivity would enable detection of small chemical variations in biological samples associated with disease. ATR-IR is particularly suitable for liquid sample analysis and, although air drying is commonly performed before data collection, just a drop of human serum is enough for screening and early diagnosis. However, the dynamic range of constituent biochemical concentrations in the serum composition remains a limiting factor to the reliability of the technique. Using glucose as a model spike in human serum, it has been demonstrated in the present study that fractionating the serum prior to spectroscopic analysis can considerably improve the precision and accuracy of quantitative models based on the Partial Least Squares Regression algorithm. By depleting the abundant high molecular weight proteins, which otherwise dominate the spectral signatures collected, the ability to monitor changes in the concentrations of the low molecular weight constituents is enhanced. The Root Mean Square Error for the Validation set (RMSEV) has been improved by a factor of 5 following human serum processing with an average relative error in the predictive values below 1% is achieved. Moreover, the approach is easily transferable to different bodily fluids, which would support the development of more efficient and suitable clinical protocols for exploration of vibrational spectroscopy based ex-vivo diagnostic tools

Imagerie IRTF tridimensionnelle pour l'étude de l'insuffisance rénale chronique

CKD (Chronic Kidney Disease) is one of the worst public diseases in developing countries. The stages of CKD are mainly based on measured or estimated GFR (Glomerular Filtration Rate). However, this method is not sensitive enough on early stages of the pathology and thus do not offer accurate diagnostic value. Early detection and treatment can often limit or avoid the chronicity effects of the disease. This thesis focuses on the development of FTIR microscopy as a diagnostic tool for the identification by histopathology at glomerulus level of the kidney in CKD model. We developed a technique of 3D reconstruction for the FTIR imaging of biochemical components changes in glomeruli for identifying the pathological marker of CKD. The curve-fitting and spectral clustering are applied on the FTIR microscopy analysis to distinguish between healthy and pathological glomeruli of a kidney. Then, the glomerular microvasculatureis highlighted to reveal the morphological abnormalities by perfusing contrast agents into blood vessels. With advanced 3D statistical methods and 3D image visualization by microscopy, FTIR spectro-imaging can be used as a functional technique to determine the morphological and molecular changes occurring along CKD development.L’insuffisance rénale chronique (IRC) et l’une des pires maladies chroniques dans les pays développés. Les grades de l’IRC sont principalement basés sur la mesure ou l’estimation du taux de filtration rénale (GFR). Cependant, cette méthode est peu sensible sur les premiers stades de la pathologie et n’apporte donc pas de valeur diagnostique. La détection de la pathologie à des stades précoces et son traitement peuvent éviter ou limiter les effets délétères de la chronicité. Cette thèse se penche sur le développement de la microscopie IRTF en tant qu’outil diagnostic pour l’identification par histopathologie à l’échelle du glomérule dans un modèle d’IRC. Nous avons développé la technique de reconstruction 3D pour l’imagerie IRTF des modifications biochimiques à l’échelle du glomérule pour déterminer des marqueurs de l’IRC. La déconvolution spectrale et le clustering sont appliqués après analyses IRTF pour distinguer les modèles sains et pathologiques. Ensuite, la microvasculature glomérulaire est révélée par agent de contraste pour en déterminer les anomalies morphologiques. Grâce aux résultats obtenus en 3D et l’utilisation de méthodes statistiques avancées, la microscopie IRTF est utilisée comme une technique fonctionnelle pour déterminer les modifications morphologiques et moléculaires apparaissant au cours du développement de l’IRC

Ischémie et angiogénèse tumorale : effets des carences en glucose et en acides aminés sur l'expression du VEGF-A par les cellules tumorales et implication de la réponse (« Unfolded Protein Response »).

Dans les tumeurs solides, les cellules soumises à des conditions ischémiques induisent des voies de signalisation distinctes contribuant à la commutation angiogénique et au développement tumoral. La composante hypoxique de l'ischémie induit l'expression du VEGF-A par la voie de signalisation dépendante de HIF-1. Par contre, la contribution des carences en glucose ou en glutamine est peu connue. En utilisant un milieu sans sérum, nous montrons que l'activation et la signalisation dépendante d'IRE1 sont des événements liant les réponses dépendantes de l'hypoxie et des carences en glucose à la surexpression du VEGF-A dans divers modèles cellulaires. Des cellules tumorales exprimant un dominant négatif de IRE1 et des cellules MEF IRE1-/- n'induisent pas l'expression du VEGF-A en conditions d'hypoxie ou de carences en glucose. Ces stress mènent à l'induction du VEGF-A par des mécanismes en partie distincts. Ces données sont corrélées avec une réduction de l'angiogenèse et de la croissance tumorale in vivo et démontrent le rôle essentiel joué par IRE1 en réponse à l'ischémie. En absence de glutamine, les ARNm du VEGF-A sont surexprimés indépendamment de l'accumulation des protéines HIF et de la transcription des gènes dépendants de la réponse UPR. De plus, l'expression protéique du VEGF-A est diminuée en corrélation avec la réduction de la synthèse protéique globale. Ces résultats suggèrent que les carences en glutamine n'ont aucun effet sur le processus angiogénique induit par le VEGF-A dans certaines tumeurs. Nous avons également recherché l'implication des carences en glutamine et en glucose dans les réponses métaboliques des cellules tumorales. Ces stress augmentent rapidement le processus d'apoptose, d'une manière plus importante en absence de glutamine qu'en absences de glucose. La spectrométrie FTIR a permis d'évaluer le statut métabolique des cellules tumorales ischémiques et a corrélé leur phénotype hyper-glycolytique avec leur statut prolifératif et agressif.In solid tumors, cells subjected to ischemic conditions trigger distinct signaling pathways which contribute to the angiogenic switch and tumor development. The hypoxic component of ischemia leads to the expression of VEGF-A by the HIF-1-dependent signaling pathways. Alternatively, the contribution of glucose or glutamine deprivation is still not well understood. Using a serum-free medium, we demonstrate that IRE1 activation and signaling is a common molecular determinant linking hypoxia- and hypoglycemia-dependent responses to the upregulation of VEGF-A in various cell models. Indeed, tumor cells expressing a dominant negative IRE1 transgene as well as IRE1-/- MEF were unable to trigger VEGF-A upregulation upon either hypoxic or glucose deprivation conditions. We showed that these various cellular stresses lead to the induction of VEGF-A by partly distinct mechanisms. These data correlated with a reduction of tumor angiogenesis and growth in vivo and demonstrated the essential role played by IRE1 in response to tumor ischemia. Under glutamine deprivation, VEGF-A mRNA was up-regulated and this effect was neither associated to HIF proteins accumulation nor to the transcriptional induction of UPR-dependent genes. Besides, VEGF-A protein expression was down-regulated in correlation to the global decrease of protein synthesis. These results suggested that amino-acids deprivation has no effect on the VEGF-A-driven angiogenic process in a number of tumors. We then questioned the implication of glucose and glutamine deprivations in the metabolic responses of tumor cells. This stress rapidly increase the apoptotic response of the cells, and to a much greater extent in the absence of glutamine than under glucose deprivation. FTIR spectrometry was used to evaluate the metabolic status of tumor cells and correlated the hyper-glycolytic phenotype with their proliferative and aggressive status

Infrared Diagnostics on Micro and Nano Scale Structures

Fourier Transform Infrared spectroscopy is used as a diagnostic tool in biological and physical sciences by characterizing the samples based on infrared light-matter interaction. In the case of biological samples, Activation of Jurkat T-cells in culture following treatment with anti-CD3 (Cluster of Differentiation 3) antibody is detectable by interrogating the treated T-cells using the Attenuated Total Reflection - Fourier Transform Infrared (ATR-FTIR) Spectroscopy technique. Cell activation was detected within 75 minutes after the cells encountered specific immunoglobulin molecules. Spectral markers noted following ligation of the CD3 receptor with anti CD3 antibody provides proof-of-concept that ATR-FTIR spectroscopy is a sensitive measure of molecular events subsequent to cells interacting with anti-CD3 Immunoglobulin G (IgG). ATR-FTIR spectroscopy is also used to screen for Colitis in chronic (Interleukin 10 knockout) and acute (Dextran Sodium Sulphate-induced) models. Arthritis (Collagen Antibody Induced Arthritis) and metabolic syndrome (Toll like receptor 5 knockout) models are also tested as controls. The marker identified as mannose uniquely screens and distinguishes the colitic from the non-colitic samples and the controls. The reference or the baseline spectrum could be the pooled and averaged spectra of non-colitic samples or the subject’s previous sample spectrum. The circular dichroism of titanium-doped silver chiral nanorod arrays grown using the glancing angle deposition (GLAD) method is investigated in the visible and near infrared ranges using transmission ellipsometry and spectroscopy. The characteristics of these circular polarization effects are strongly influenced by the morphology of the deposited arrays. Studies of optical phonon modes in nearly defect-free GaN nanowires embedded with intrinsic InGaN quantum dots by using oblique angle transmission infrared spectroscopy is described here. These phonon modes are dependent on the nanowire fill-factor, doping densities of the nanowires and the presence of InGaN dots. These factors can be applied for potential phonon based photodetectors whose spectral responses can be tailored by varying a combination of these three parameters. The optical anisotropy along the growth (c-) axis of the GaN nanowire contributes to the polarization agility of such potential photodetectors

A study of FT-IR spectroscopy for the identification and classifcation of haematological malignancies

The aim of the work presented in this thesis was to explore the use of FT-IR spectroscopy, as a complementary clinical tool for haematological laboratory analysis. FT-IR spectra were measured from air-dried and frozen cell lines derived from lymphoma, lymphoid, myeloid leukaemia and normal and chronic lymphocytic leukaemia blood samples. Multivariate statistical analysis was used to extract important spectral information with the greatest discriminative power. Principal component fed linear discriminant spectral models have been tested with leave one out cross validation procedures. A preliminary unfiltered classification model using 50 frozen and air-dried samples correctly classified 54% of 18556 spectra. The performance improved with the three cell line group datasets, with 71% of 19903 spectra correctly classified. Furthermore, the use of the frozen spectra improved the performance of the three cell line group classification model considerably. Findings showed that 73.3% of 9920 spectra were correctly classified in the frozen datasets, whereas in the air-dried only 41.5% of 9983 spectra are correctly classified. Optimisation of the spectral models by selection of principal components, application of Savitsky-Golay filters and selecting spectra using standard deviation and absorption filter tool was investigated. Using the first 25 significant PCs, a 0 th derivative Savitsky-Golay filter and the absorbance filter tool on the frozen five cell line spectral dataset were shown to be the optimal parameters for constructing a classification model. When tested with leave one batch out cross validation 90% of the spectra were correctly classified for the five cell line model. Blood component classification models tested with leave one batch out cross validation performed well. The whole blood model correctly classified 70% of 1736 spectra, measured on 22 samples. The plasma model correctly classified 80.6% of 331 spectra and the buffy coat model correctly classified 99.5% of 1438 spectra. This demonstrated that the buffy coat (containing white blood cells) holds the key biochemical information for discrimination between the pathology of the blood samples. Partial least squares analysis has been demonstrated as a method to support whole blood count tests for real time prediction of cellular constituents. These findings demonstrate the potential of FT- IR spectroscopy as a clinical tool although more work is needed if it is to be applied in clinical practice.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Identificação de biomarcadores de Alzheimer por FTIR: estudo de caso

Mestrado em Biomedicina MolecularThe absorption of radiation in the mid-infrared spectral region provides detailed information on the chemical composition of biological samples. Most molecular species absorbs infrared light, giving rise to distinctive spectral patterns in transmitted light. The pathological conditions are associated with metabolic disorders that are reflected in homeostatic changes of molecular components of cells and tissues. The clinical potential of Fourier Transform Infrared (FTIR) spectroscopy to detect such changes and its use as a diagnostic tool has received increasing attention. This technique allows the identification of signals associated with dementia at a biochemical level, and helps to identify specific biomarkers. This dissertation is an exploratory study, which aimed to test the ability of FTIR to discriminate between control and Alzheimer’s Disease (AD) samples and to identify spectroscopic signals spectroscopic signals corresponding to functional groups of biomarkers present in serum and plasma of the disease. Blood samples were collected and volunteers of the study were submitted to cognitive evaluation through Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR). Relevant clinical information was also collected. Samples of putative Alzheimer's patients, as well as samples of the controls matched by age and sex were analyzed by FTIR. Spectra in the range 4000-900 cm-1 were submitted to Principal Component Analysis (PCA). With the diagram of the factorial coordinates (scores) of the PCA was possible to discriminate the sample spectra of putative Alzheimer's patients, of the sample spectra of controls. The diagram of the factorial contributions (loadings) allowed the identification of spectroscopic signals associated with functional groups of biomarkers involved in discrimination between samples from patients and controls. In future, after matching all spectroscopic signals of the corresponding AD biomarkers and gathering biomarker (s) responsible for the spectroscopic identification of AD will be possible to develop a model of multivariate classification.A absorção de radiação na região espectral do infravermelho médio fornece informações detalhadas sobre a composição química das amostras biológicas. A maioria das espécies moleculares absorve a luz infravermelha, que dá origem a padrões espectrais característicos na luz transmitida. As condições patológicas estão associadas a perturbações do metabolismo, que se refletem em alterações homeostáticas dos componentes moleculares das células e dos tecidos. O potencial clínico da espectroscopia de infravermelho por transformada de Fourier (FTIR) para detetar tais mudanças e o seu uso como uma ferramenta de diagnóstico tem recebido cada vez mais atenção. Esta técnica permite a identificação de sinais associados à demência, a um nível bioquímico, e auxilia na identificação de biomarcadores específicos. Esta dissertação é um estudo exploratório que pretendeu testar a capacidade da espectroscopia FTIR para discriminar amostras de indivíduos controlo de amostras de possíveis doentes de Alzheimer, e identificar sinais espectroscópicos correspondentes a grupos funcionais de biomarcadores presentes no soro e no plasma relativos à doença. As amostras de sangue foram colhidas e os voluntários do estudo foram submetidos à avaliação cognitiva através do Mini Exame do Estado Mental (MEEM) e da Escala de Avaliação Clínica da Demência (CDR). Informações clínicas relevantes também foram recolhidas. As amostras dos possíveis doentes de Alzheimer, assim como as amostras dos controlos pareados com a mesma idade e sexo foram analisadas por FTIR. Os espectros na gama de 4000-900 cm-1 foram submetidos à análise em componentes principais (ACP). Com o diagrama das coordenadas fatoriais (scores) do PCA foi possível discriminar os espectros das amostras de possíveis doentes de Alzheimer dos espectros das amostras dos controlos. O diagrama das contribuições fatoriais (loadings) permitiu a identificação de sinais espectroscópicos associados a grupos funcionais de biomarcadores envolvidos na discriminação entre amostras de doentes e controlos. Futuramente, após a recolha de todos os sinais espectroscópicos correspondentes aos biomarcadores de AD e de definir o (s) biomarcadores responsável (eis) pela identificação espectroscópica de AD, será possível desenvolver um modelo de classificação multivariada