Thermo-stabilized lignin fibers from pH-fractionated softwood kraft lignin were carbonized to various temperatures during thermomechanical analysis (TMA) under static and increasing load and different rates of heating. The aim was to optimize the carbonization process to obtain suitable carbon fiber material with good mechanical strength potential (high tensile strength and high E-modulus). The carbon fibers were therefore mainly evaluated of mechanical strength in Dia-Stron uniaxial tensile testing. In addition, chemical composition, in terms of functional groups, and elemental (atomic) composition was studied in Fourier transform infrared spectroscopy (FTIR) and in energy-dispersive X-ray spectroscopy (EDS), respectively. The structure of carbon fibers was imaged in scanning electron microscope (SEM) and light microscopy. Thermogravimetrical analysis was performed on thermo-stabilized lignin fibers to evaluate the loss of mass and to calculate the stress-changes and diameter-changes that occur during carbonization. The TMA-analysis of the deformation showed, for thermo-stabilized lignin fibers, a characteristic behavior of contraction during carbonization. Carbonization temperatures above 1000°C seemed most efficient in terms of E-modulus and tensile strength whereas rate of heating did not matter considerably. The E-modulus for the fibers was improved significantly by slowly increasing the load during the carbonization. The tensile strength remained however unchanged. The FTIR-analysis indicated that many functional groups, mainly oxygen containing, dissociate from the lignin polymers during carbonization. The EDS supported this by showing that the oxygen content decreased. Accordingly, the relative carbon content increased passively to around 90% at 1000°C. Aromatic structures in the carbon fibers are thought to contribute to the mechanical strength and are likely formed during the carbonization. However, the FTIR result showed no evident signs that aromatic structures had been formed, possible due to some difficulties with the KBr-method. In the SEM and light microscopy imaging one could observe that porous formations on the surface of the fibers increased as the temperature increased in the carbonization. These formations may have affected the mechanical strength of the carbon fibers, mainly tensile strength. The carbonization process was optimized in the sense that any heating rate can be used. No restriction in production speed exists. The carbonization should be run to at least 1000°C to achieve maximum mechanical strength, both in E-modulus and tensile strength. To improve the E-modulus further, a slowly increasing load can be applied to the lignin fibers during carbonization. The earlier the force is applied, to counteract the lignin fiber contraction that occurs (namely around 300°C), the better. However, in terms of mechanical performance, the lignin carbon fibers are still far from practical use in the industry.

A conventional membrane bioreactor (MBR) and two moving bed bioreactors coupled with ultrafiltration membrane filtration were operated for close to six months to investigate biological nutrient removal and potential fouling inducing parameter mitigation. Unique to one of the moving bed membrane bioreactors (MBMBR) was a newly designed media that incorporated a hydrodynamic exterior carrier with a highly porous interior packing. Preliminary investigation indicates that nitrogen compounds were superiorly removed in the two MBMBRs when compared with the MBR. This is a result of denitrification processes occurring in anoxic micro-zones found within the depths of the biofilm affixed to media. Fouling propensity was found to be increased by over four times in the MBMBR systems as compared to the MBR. Mixed liquor, permeate and filtrate analysis, membrane fibre examination and permeability tests indicated that colloidal organic carbon, as well as soluble microbial products were the dominant fouling inducing compounds. / Manuscript format / The Natural Sciences and Engineering Research Council of Canada

Thesis (MSc Food Sc )--Stellenbosch University, 2000. / ENGLISH ABSTRACT: This study shows Fourier transform near infrared (FT-NIR) spectroscopy's application on wine, fruit and dried fruit for quantitative determinations or as a discriminative method for classification purposes. During wine production optimum yeast growth, resulting in healthy alcohol fermentation rates, is monitored by the amount of free amino nitrogen (FAN) present in the must. The status of malolactic fermentation (MLF) in Chardonnay wines is monitored by determining the degree of conversion of malic to lactic acid. Ethyl carbamate (EC), a suspected carcinogen, is mainly formed during ageing of wine and is restricted by legislation in some countries. It is therefore necessary to determine the EC content in wine. Fourier transform near infrared spectroscopy can be used on wine as a rapid method to measure the °Brix (residual sugars) content of must and to discriminate between different must samples in terms of their FAN values. It can also be used as a rapid method to discriminate between Chardonnay wine samples in terms of the MLF status and between table wine samples in terms of the EC content. Calibrations were derived and it was found that a very strong correlation existed in the sample set for the FT-NIR spectroscopic predictions for °Brix (r = 0.99, SEP = 0.31%), but poorer correlations for the FAN (r = 0.405, SEP = 275%), malic acid (r = 0.64, SEP = 1.02%), lactic acid (r = 0.61, SEP = 1.35%) and EC predictions (r = 0.47, SEP = 3.6%). When soft independent modelling by class analogy (SIMCA) was applied as a discriminative method, the must and wine samples were classified in terms of their FAN and EC values and MLF status, respectively, obtaining results with recognition rates exceeding 80%. The canning of peaches has become a significant industry in South Africa, producing approximately 4.1 million cartons per year. Fourier transform near infrared spectroscopy was applied as an alternative non-destructive method for the quantitative determination of the total soluble solid (TSS) content of whole fresh peaches. The TSS content of fresh clingstone peaches is an indication of the internal quality, maturity and perceived sweetness of the fruit for the peach canning industry. By determining the TSS, fresh peaches can be graded and the farmers compensated accordingly. Results obtained by building QUANT+™calibrations for the TSS content (r = 0.96, SEP = 0.55%) showed acceptable accuracy and can replace the present destructive methods. Peak periods during the harvesting season necessitate storage of peaches for up to three weeks before canning. Approximately 5 - 10% of the peaches stored, disintegrate during canning due to loose skin, large stone cavities, soft flesh and rot. The storage potential of fresh clingstone peaches can be successfully predicted with FT-NIR and SIMCA models, using subjective internal quality evaluations. Results with recognition rates exceeding 80% were obtained in most cases and this method proved useful as a non-destructive method of quality assessment. By applying this method, losses caused when storing peaches with poor storage quality will be reduced. The golden sultana industry plays an important role in the dried fruit exporting market of South Africa. Due to the large numbers of consignments that must be checked upon arrival, and the need for rapid decision making during processing, it is essential to replace the present time-consuming analytical methods. Fourier transform near infrared spectroscopy was used as a rapid, analytical technique to determine whether the S0₂ and moisture contents of sultanas are within specifications upon arrival at the factory and during processing. High positive correlation was found between the measured values and those predicted by FT-NIR spectroscopy for S0₂ (r = 0.99, SEP = 24.09%) and moisture (r = 0.99, SEP = 0.051 %) contents. / AFRIKAANSE OPSOMMING: Hierdie studie dui op Fourier transformasie naby-infrarooi (FT-NIR) spektroskopie se toepassing op wyn, vrugte en droëvrugte vir die uitvoer van kwantitatiewe bepalings of vir klassifikasie doeleindes om as 'n diskriminasie metode te dien. Gedurende wynproduksie word die optimum groei van giste wat lei tot 'n gesonde alkohol fermentasie gemonitor deur die hoeveelheid vry-aminostikstof (VAS) wat in die mos teenwoordig is te bepaal. Die status van appelmelksuurgisting (AMG) in Chardonnay wyne word gemonitor deur die mate van omskakeling van appelsuur na melksuur te bepaal. Etielkarbamaat (EK), 'n vermoede karsinogeen wat hoofsaaklik in verouderde wyne voorkom, word in sekere lande deur wetgewing beperk en dus die bepaling van die EK inhoud van wyne noodsaak. Fourier transformasie naby-infrarooi spektroskopie kan op mos toegepas word as 'n vinnige metode vir die bepaling van °Brix (residuele suiker) en om tussen die monsters te onderskei in terme van hulle VAS inhoud. FT-NIR kan ook gebuik word as 'n vinnige metode om tussen Chardonnay monsters te onderskei op grond van die status van AMG en tussen tafelwyn monsters op grond van die EK inhoud. Kalibrasies is ontwikkel en daar is gevind dat baie sterk korrelasies bestaan in die monsterstel vir die FT-NIR spektroskopiese voorspelling van °Brix (r = 0.99, SEP = 0.31%), maar swakker korrelasies vir die VAS (r = 0.405, SEP = 275%), appelsuur (r = 0.64, SEP = 1.02%), melksuur (r = 0.61, SEP = 1.35%) en EK voorspellings (r = 0.47, SEP = 3.6%). Met die toepassing van soft independent modelling by class analogy (SIMCA) as diskriminasie metode, is die mos- en wynmonsters geklassifiseer op grond van hul VAS en EK waardes en die status van AMG, en herkenningswaardes van bo 80% is onderskeidelik behaal. Die inmaak van perskes het 'n beduidende industrie in Suid-Afrika geword en produseer jaarliks ongeveer 4.1 miljoen kartonne. Fourier transformasie nabyinfrarooi spektroskopie is toegepas as alternatiewe, nie-beskadigende metode om kwantitatiewe bepalings van die totale oplosbare vastestowwe (TOV) inhoud van heel vars perskes, te doen. Vir die perske inmaak-industrie is die TOV inhoud van vars taaipitperskes 'n aanduiding van interne kwaliteit, rypheid en die soetheid van die vrugte. Vars perskes kan gradeer word deur die TOS te bepaal en sodoende kan boere oreenkomstige vergoeding ontvang. Resultate' wat verkry is deur QUANT+™ kalibrasies vir TOS inhoud te ontwikkel (r = 0.96, SEP = 0.55%), dui op aanvaarbare akkuraatheid en kan die huidige metodes vervang. Tydens oestyd kom piektye voor wanneer dit soms nodig is om perskes vir tot drie weke op te berg voordat dit ingemaak kan word. Ongeveer 5 tot 15% van hierdie opgebergte perskes disintegreer tydens inmaak omdat opberging lei tot defekte in die perskes soos skille wat loskom, groot pitholtes, sagte vleis en vrot. Die opbergingspotentiaal van vars taaipitperskes kan suksesvol voorspel word deur FTNIR en SIMCA modelle te bou en subjektiewe interne kwaliteitsevaluerings daarop toe te pas. Herkenningsresultate wat 80% in die meeste gevalle oorskry, is behaal wat hierdie metode as 'n suksesvolle nie-beskadigende kwaliteitbepalingsmetode bewys. Hierdie metode sal verliese kan beperk wat voorkom as gevolg van opberging van perskes met swak opbergingskwaliteit. Die goue sultana industrie speel 'n belangrike rol in die droë vrugte uitvoermark van Suid-Afrika. As gevolg van die hoeveelheid monsters wat gelyktydig getoets moet word en besluite wat vinnig geneem moet word tydens prosessering, is dit belangrik om die huidige tydrowende analitiese metodes te vervang. Fourier transformasie naby-infrarooi spektroskopie is gebruik as 'n vinnige, analitiese tegniek om tydens ontvangs by die fabriek en gedurende prosessering te bepaal of die S0₂- en voginhoud van goue sultanas binne die spesifikasies val. Goeie positiewe korrelasie is gevind tussen die bepaalde en voorspelde FT-NIR spektroskopiese waardes vir SO₂- (r = 0.99, SEP = 24.09%) en voginhoud (r = 0.99, SEP = 0.051 %).

Formaldehyde-based adhesives have been reported to be detrimental to health. Petrochemical-based adhesives are non-renewable, limited and costly. Therefore, the improvement of environmental-friendly adhesive from natural agricultural products has awakened noteworthy attention. A novel adhesive for wood application was successfully prepared with enhanced shear strength and water resistance. The Fourier transmform infrared spectra showed the surface functionalities of the functionalized carbon nanotubes (FCNTs) and soy protein isolate nanocomposite adhesive. The attachment of carboxylic functional group on the surface of the carbon nanotubes (CNTs) after purification contributed to the effective dispersion of the CNTs in the nanocomposite adhesive. Hence, enhanced properties of FCNTs were successfully transferred into the SPI/CNTs nanocomposite adhesive. These unique functionalities on FCNTs however, improved the mechanical properties of the adhesive. The shear strength and water resistance of SPI/FCNTs was higher than that of the SPI/CNTs. SEM images showed the homogenous dispersion of CNTs in the SPI/CNTs nanocomposite adhesive. The carbon nanotubes were distributed uniformly in the soy protein adhesive with no noticeable clusters at relatively reduced fractions of CNTs as shown in the SEM images, which resulted into better adhesion on wood surface. Mechanical (shear) mixing and ultrasonication with 30 minutes of shear mixing both showed an improved dispersion of CNTs in the soy protein matrix. However, ultrasonication method of dispersion showed higher tensile shear strength and water resistance than in mechanical (shear) mixing method. Thermogravimetric analysis of the samples also showed that the CNTs incorporated increases the thermal stability of the nanocomposite adhesive at higher loading fraction. Incorporation of CNTs into soy protein isolate adhesive improved both the shear strength and water resistance of the adhesive prepared at a relatively reduced concentration of 0.3%.The result showed that tensile shear strength of SPI/FCNTs adhesive was 0.8 MPa and 7.25MPa at dry and wet state respectively, while SPI/CNTs adhesive had 6.91 MPa and 5.48MPa at dry and wet state respectively. There was over 100% increase in shear strength both at dry and wet state compared to the pure SPI adhesive. The 19% decrease in value of the new adhesive developed compared to the minimum value of ≥10MPa of European standard for interior wood application may be attributed to the presence of metallic particles remaining after purification of CNTs. The presence of metallic particles will prevent the proper penetration of the adhesive into the wood substrate. The type of wood used in this study as well as the processing parameters could also result into lower value compared to the value of European standard. Therefore, optimization of the processing parameter as well as the conversion of carboxylic acid group on the surface of the CNTs into acyl chloride group may be employed in future investigation. However, the preparation of new nanocomposite adhesive from soy protein isolate will replace the formaldehyde and petrochemical adhesive in the market and be of useful application in the wood industry. / Civil and Chemical Engineering / M. Tech. (Chemical Engineering)

Les arbres sont des organismes de grande longévité qui se développent dans un environnement variable. Au cours de leur formation, ils génèrent une tension appelée contrainte de maturation pour remplir des fonctions biomécaniques essentielles. Chez les angiospermes, les arbres adaptent leur état mécanique par la production de bois de tension avec de fortes contraintes de traction sur la face supérieure de la tige penchée. Malgré les recherches considérables dans ce domaine durant de nombreuses années, les connaissances actuelles sur le mécanisme de la génération active de contrainte dans le bois de tension sont encore incomplètes et doivent être améliorées. La première partie de cette étude était de faire progresser la compréhension de la composition et de l'organisation des polymères de la paroi cellulaire secondaire, ainsi que leur orientation au cours de la maturation de la paroi cellulaire du bois de tension. Les mesures effectuées sur la microscopie FTIR ont indiqué qu'avant même la formation de la couche G, il existait une structure d'hydrates de carbone ordonnée à un angle plus parallèle à l'axe de la fibre dans le bois de tension. Ces résultats étaient clairement différents du comportement du bois opposé. Dans le bois de tension, la lignine était plus fortement orientée dans la couche S2 que dans le bois opposé. Avec la formation de la couche S2 dans le bois opposé et de la couche G dans le bois de tension, les signaux d'orientation des hydrates de carbone amorphes tels que les hémicelluloses et les pectines sont différents entre le bois de tension et le bois qui lui est opposé. Pour les bois de tension, l'orientation de ces bandes est la même tout au long du processus de maturation de la paroi cellulaire, ce qui reflète probablement un dépôt continu de xyloglucane ou de xylane, avec une orientation différente de celle de la paroi S2 pendant tout le processus. La seconde partie de ce travail était d'améliorer les connaissances actuelles sur le comportement de la matrice par l'étude de la mésoporosité et de son évolution lors de la construction et de la maturation de la paroi cellulaire du bois de tension. Les résultats sur deux types de bois de tension suggèrent que la mésoporosité peut toujours être détectée près de la zone de cambium autant pour le bois de tension que pour le bois opposé. La forte porosité diminue progressivement avec la lignification dans la paroi cellulaire en développement, avec une exception pour le bois de tension à couche G. La porosité de type bouteille d'encre et l'augmentation de la taille médiane des pores sont observées dans les deux types de bois de tension, indiquant que les espèces de bois de tension avec et sans couches G peuvent partager le même mécanisme de génération de contrainte de traction. Cette étude contribue à une meilleure compréhension de la génération de contrainte de maturation dans les arbres et peut servir de base pour l'amélioration de la modélisation du comportement de la matrice au cours de la maturation de la paroi cellulaire. / Trees are long-living organisms which develop in a variable environment. During their formation, they generate a tensile mechanical stress called maturation stress to fulfil essential biomechanical functions. In angiosperm species, trees adapt the mechanical state by producing tension wood with high tensile stresses on the upper side of the leaning stem. Despite considerable research in this field during a number of years, the current knowledge on the mechanism of the active stress generation in tension wood is still incomplete and needs improvement. The first part of this study was to advance the understanding on the composition and organisation of polymers within the secondary cell wall, as well as its orientation during the maturation of tension wood cell wall. Measurements performed on FTIR microscopy indicated that already before G-layer formation, a more ordered structure of carbohydrates at an angle more parallel to the fibre axis exists in tension wood. This was clearly different to the behaviour of opposite wood. In tension wood, the lignin was more highly oriented in the S2 layer than in opposite wood. With the formation of the S2 layer in opposite wood and the G-layer in tension wood, the orientation signals from the amorphous carbohydrates like hemicelluloses and pectins were different between opposite wood and tension wood. For tension wood, the orientation for these bands remains the same all along the cell wall maturation process, probably reflecting a continued deposition of xyloglucan or xylan, with an orientation different to that in the S2 wall, throughout the whole process. The second part of this study was to improve the current knowledge on the matrix behaviour by studying the mesoporosity and its evolution during the building and maturation of tension wood cell wall. Results on two kinds of tension wood suggested that mesoporosity can always be detected near cambium zone for both tension and opposite wood. The high porosity decreased gradually with the lignification in the developing cell wall, with an exception in tension wood with G-layer. The typical ink-bottle pore and the increase of median pore size are observed in both kinds of tension wood, indicating non-G-layer species may share the same mechanism of tensile stress generation as in tension wood with G-layer. This study aims to contribute to an increased understanding on the maturation stress generation in trees and may allow to improve the modelling of matrix behaviour during cell wall maturation.

Resumo: O objetivo deste trabalho, dividido em dois estudos, foi avaliar: (1) a influência dos tipos de ponteiras (fibra óptica e polímero) utilizadas em um aparelho fotoativador à base de LED (Ultrablue IS - DMC) sobre o grau de conversão e dureza de uma resina composta microhíbrida (FiltekTM Z250) e uma nanoparticulada (FiltekTM Supreme XT); e (2) a influência dos tipos de ponteiras (fibra óptica e polímero) utilizadas em um aparelho fotoativador à base de LED (Ultrablue IS - DMC) sobre a resistência à compressão de uma resina composta microhíbrida (FiltekTM Z250) e uma nanoparticulada (FiltekTM Supreme XT). No primeiro estudo, para os testes de grau de conversão (GC) e dureza, cinco corpos-de-prova com 4 mm de diâmetro e 2 mm de espessura (ISO 4049), foram confeccionados para cada grupo avaliado. O grau de conversão foi analisado pelo Espectrofotômetro Nexus - 470 FT-IR. Para o teste de dureza Vickers, os corpos-de-prova foram levados ao Durômetro Micromet 2100 (Buehler, EUA) onde foi utilizada uma carga de 50 gramas força (gf) e tempo de 30 segundos. Para cada corpo-de-prova oito medidas foram realizadas nas superfícies de topo e base. Os dados obtidos para o GC e dureza foram analisados estatisticamente pelo teste de Análise de Variância (ANOVA) e Tukey. No segundo estudo, para o teste de resistência à compressão foram confeccionados oito corpos-de-prova com 8 mm de altura e 4 mm de diâmetro. O teste foi realizado na máquina de ensaio universal EMIC com célula de carga de 5 kN à velocidade de 0,5 mm/min. Os dados obtidos foram analisados estatisticamente pelo teste ANOVA e o teste Tamhane. Os resultados demonstraram que o GC e dureza foram influenciados pelas ponteiras, sendo a dureza também influenciada pelo tipo de resina composta utilizada. A resina composta microhíbrida fotoativada com a ponteira de fibra óptica apresentou maiores valores do GC e dureza... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this work, divided into two studies, was evaluate: (1) the influence of types of light guide tips (fiber optic and polymer) used in a photo-activation based on LED (Ultrablue IS - DMC) on the degree of conversion and hardness one microhybrid FiltekTM Z250 and one nanofilled FiltekTM Supreme XT composite resins; and (2) the influence of types of light guide tips (fiber optic and polymer) used in a photo-activation based on LED (Ultrablue IS - DMC) on the compressive strength of one microhybrid FiltekTM Z250 and one nanofilled FiltekTM Supreme XT composite resins. In the first study, to test the degree of conversion (GC) and hardness, five samples 4 mm in diameter and 2 mm in thickness (ISO 4049) were made for each group evaluated. The degree of conversion was evaluated by Fourier transform infrared spectroscopy (FT-IR). For the Vickers hardness test, the samples were brought to the durometer Micromet 2100 (Buehler, USA) where was used a load of 50 gram force (gf) and time of 30 seconds. For each sample test measurements were performed eigth measures on the surfaces of top and bottom. The data obtained for the GC and hardness test were statistically analyzed by Analysis of Variance (ANOVA) and Tukey's test. In the second study to test the compressive strength eight samples (4 mm in diameter and 8 mm in thickness) were made for each group evaluated, and the test was conducted in a universal testing machine EMIC with load cell 5 kN and speed of 0,5 mm/min. The data were statistically analyzed by ANOVA test and Tamhane's test. The results showed that the GC and hardness were influenced by the light guide tip, and the hardness also influenced by the type of resin used. The microhybrid composite resin photo-activated with the fiber optic light tip had higher values of GC and hardness. The lowest values of GC and hardness were observed with nanofilled composite resin photoactivated... (Complete abstract click electronic access below) / Orientador: Marcelo Ferrarezi de Andrade / Coorientador: Alessandro Nara de Souza Rastelli / Banca: Silmara Aparecida Milori Corona / Banca: José Roberto Cury Saad / Mestre

An early diagnosis of melanoma is essential to reduce mortality of patients. The diagnosis is also fundamental to predict the outcome of patients and to select the most adapted treatment. Current diagnostic assessments are obtained after visual inspection of the histological section of the primary tumor. The pathologist has first to determine the malignant nature of the lesion and then to assess the potential of the lesion to form metastases. Depending on several characteristics of the primary tumor (mainly tumor thickness, ulceration and mitotic rate), the sentinel node is surgically removed and the detection of tumor cells is based on its histopathological examination. These assessments are time consuming, to some degree subjective and are particularly challenging. Among melanoma patients that are not subject to sentinel node surgery, 6.5 % will develop metastases while 20 % of patients that undergo sentinel node surgery will effectively present metastases. The search for biomarkers that can identify malignant cells, evaluate potential of invasion or help selecting a treatment is still on.<p>In this thesis we used a new and promising technique of imaging based on infrared spectroscopy to study melanoma primary tumors and metastatic lymph nodes. Infrared spectroscopy brings information on the biochemical composition of the main components of the cells. When combined with a microscope and with multivariate statistical analyses, images that are generated allow the identification of melanoma cells and stromal cells in the biopsy. We also focused on the immune infiltration as it was shown to carry an important prognosis value for melanoma patients.<p>The first part of the thesis was a prerequisite for the rest of the study. It addresses the effects of the process of fixation that tissues obtained by surgical resection undergo for their long term preservation. In chapter III, we showed that Formalin-Fixation and Paraffin-Embedding (FFPE) procedure induces small but significant modifications in the infrared spectra of cells but these are very similar for different cell lines. In turn, it preserves the potential to identify closely-related cell lines by infrared spectroscopy.<p>We thus pursued our study on primary melanomas. In chapter IV, we first developed an automatic tool capable of identifying melanoma cells and the main cells of the tumor microenvironment in tissue sections. Importantly, we built a second model that brings information on the presence of metastases on the basis of the spectral signature of the primary tumor.<p>The next chapter is dedicated to the prediction of the response of melanoma to dacarbazine, the first-line chemotherapy to treat stage IV patients. Infrared spectra of the primary tumor were shown to contain information capable of predicting whether dacarbazine will be a useful treatment.<p>In the last two chapters, we focused on lymphocytes. In chapter VI, we first demonstrated that helper and cytotoxic T cells purified from peripheral blood can be identified on the basis of their infrared signature. Then, in chapter VII we investigated metastatic lymph nodes. We created different statistical models using infrared spectra that first identified the melanoma cells invading the lymph nodes and secondly, the different subpopulations of lymphocytes (B and T cells).<p>In conclusion, we developed an automatic and reliable tool of imaging to help pathologists in the anatomopathological assessment of primary lesions and lymph nodes./Le mélanome est la forme de cancer cutané la plus mortelle, provoquant environ 80% des décès dus à un cancer de la peau. Lorsque le mélanome est localisé, la chirurgie est le principal traitement et est suffisante pour 80% des patients. A l’opposé, lorsque le mélanome primaire a formé des métastases, le cancer devient beaucoup plus difficile à traiter et la survie de ces patients diminue drastiquement. Seuls 10% des patients vont vivre 5 ans lorsqu’ils développent des métastases à distance. C’est pourquoi un diagnostic précoce est essentiel pour diminuer la mortalité causée par le mélanome. L’étape du diagnostic est également très importante pour donner un pronostic et pour planifier les traitements. Le diagnostic actuel du mélanome est basé sur l’analyse en microscopie optique de sections de la tumeur primaire. Sur base de la morphologie cellulaire et de l’architecture du tissu, cette étape permet premièrement d’identifier le caractère cancéreux de la lésion et deuxièmement d’évaluer son potentiel métastatique. Une analyse du ganglion sentinelle permet également de détecter la présence de métastases. Dans le cadre de cette thèse, nous proposons d’utiliser une nouvelle technique d’imagerie basée sur la spectroscopie infrarouge qui apporte une information complète et unique sur la biochimie de la cellule et des tissus. Les résultats présentés ici indiquent que lorsque les spectres infrarouges sont combinés à des analyses statistiques multivariées, des images sont reconstituées et révèlent les structures et les composants cellulaires majeurs présents dans les coupes de mélanomes. Une étude préliminaire a d’abord pu démontrer que la fixation au formol subies par les biopsies pour les conserver n’entrave pas l’étude de celles-ci par spectroscopie infrarouge. Nous nous sommes alors intéressés aux tumeurs primaires de mélanome et avons développé un modèle statistique, à partir des spectres infrarouges, identifiant automatiquement les cellules de mélanome dans le tissu ainsi que les autres cellules du microenvironnement de la tumeur. Dans ce chapitre, nous avons également créé un autre modèle statistique capable de prédire le potentiel métastatique de la tumeur primaire en se basant sur sa signature spectrale. Nous avons ensuite mis en évidence une signature spectrale corrélée à la réponse à la dacarbazine chez des patients traités pour leurs métastases. Nous avons également montré que des sous-populations de lymphocytes purifiées d’échantillons sanguins pouvaient être identifiées sur base de leur spectre. Cette capacité de la spectroscopie infrarouge à distinguer les différents types de lymphocytes a ensuite été démontrée pour les lymphocytes infiltrant les métastases. Finalement, nous avons mis en évidence l’utilité de cette technique d’imagerie pour la détection de métastases ganglionnaires.<p> / Doctorat en sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

O objetivo do presente trabalho foi avaliar diferentes tratamentos na remineralização do esmalte desmineralizado em situação de alto desafio cariogênico. Foram utilizados 55 fragmentos de esmalte bovino (4mmX4mmX2mm) planificados e polidos. Os espécimes foram cobertos com resina composta ficando exposta apenas a metade da superfície externa (2mmX4mm) e então, foram imersos em solução tampão de acetato (pH 4,7) por 43 horas para formar a lesão de desmineralização. Os espécimes foram divididos aleatoriamente em 5 grupos (n=11), de acordo com os tratamentos preventivos: 1- Controle (sem tratamento); 2- Verniz fluoretado a 5% Duraphat®; 3- Flúor-gel 1,23%; 4- MI PasteTM; e 5- MI Paste PlusTM. Os tratamentos foram realizados uma vez por semana durante 3 semanas. Nos intervalos das aplicações os espécimes foram submetidos a ciclos de pH (DES/RE) num total de 21 dias. Após a finalização da ciclagem os fragmentos foram seccionados longitudinalmente, sendo que em uma metade foi analisada a microdureza longitudinal, pelo cálculo da variação percentual da dureza e na outra metade foi avaliada a profundidade de desmineralização do esmalte através de microscopia óptica. Para a análise em FTIR, pó do esmalte foi obtido dos espécimes em três diferentes momentos: inicial sem desmineralização da superfície, após a desmineralização por 43 horas e após o tratamento. Para análise da microdureza longitudinal empregou-se a variação da dureza. Os dados foram analisados quanto a sua distribuição, porém como não foram normais realizou-se uma análise não paramétrica comparando grupo contra grupo pelo Mann Whitney test com &alpha;=5%. Para análise da profundidade de desmineralização, como os dados se apresentaram normais e homogêneos, realizou-se a análise de variância a 1 critério e teste Holm- Sidak para comparação das médias com &alpha;=5%. Para análise em FTIR fez-se apenas uma análise descritiva dos dados. Na análise dos dados pode-se observar que houve diferença estatística significante entre o grupo controle (G1) com o grupo flúor-gel (G3) (p<0,05), o qual apresentou menor perda de dureza e menor profundidade de desmineralização. Os grupos 2, 4 e 5 foram similares entre si e com o G1 para as duas propriedades analisadas. Na análise da absorbância nos diferentes comprimentos de onda pode-se observar um padrão similar em todas as amostras; a banda de fosfato inicial foi mais larga e posicionada em número de ondas menores. Após a desmineralização, esta banda ficou mais estreita e deslocada para número de ondas maiores. Esse comportamento se repetiu e foi similar para todos os grupo. Baseado nos resultados obtidos pode-se concluir que nenhum dos tratamentos preventivos realizados foi capaz de inibir o processo de desmineralização ou promover a remineralização em situações de alto risco a cárie, embora o flúor-gel tenha permitido uma evolução mais lenta da desmineralização / The aim of this study was to evaluate different treatments on remineralization of demineralized enamel in a high cariogenic challenge. 55 enamel fragments (4mmX4mmX2mm) flat and polished were used. The specimens were covered with composite exposed only half of the outer surface (2mmX4mm) and then were immersed in acetate buffer solution (pH 4.7) for 43 hours to form the demineralization lesion. The specimens were randomly divided into 5 groups (n = 11) according with the preventive treatments: 1 - Control (no treatment); 2 - 5% fluoride varnish Duraphat ®; 3 - Fluoride-gel 1.23%; 4 - MI PasteTM; and 5 - MI Paste PlusTM. The treatments were carried out once a week for 3 weeks. During intervals and among applications the specimens were subjected to pH cycles (DES / RE) for a total of 21 days. After finished of the cycling, the samples were longitudinally sectioned, and one half in the longitudinal microhardness was analyzed and the other half the depth of enamel demineralization using optical microscopy was evaluated. For data analyzed was calculate the percentage change in the hardness. For FTIR analysis, the enamel powder was obtained from specimens at three different times: without demineralization of the surface, after demineralization for 43 hours and after treatment. Data were analyzed for their distribution, but they were not normal there was a non-parametric analysis comparing group versus group by t test (Mann- Whitney test) with &alpha;= 5%. To analyze the depth of demineralization, as the data were normal and homogeneous, we performed analysis of variance criterion 1 and Holm-Sidak test for comparison of means with &alpha;= 5%. For FTIR was realized of descriptive analysis of the data. Data analyzed showed statistically significant difference between the control group (G1) with the fluoride gel group (G3) (p <0.05), which showed less loss of lower hardness and depth of demineralization. Groups 2, 4 and 5 were similar among them and with G1 for two properties analyzed. The analysis of the absorbance at different wavelengths can be observed a similar pattern in all samples; band initial phosphate was wider and placed on the number of smaller waves. After demineralization, this band became narrower and shifted to higher wave number. This behavior was similar for all groups and it is repeated for every group. Based on the results this study it can be concluded that none of the preventive treatment carried out was able to inhibit the demineralization or promote remineralization in high caries risk situations, although fluoride gel has allowed a slow evolution of demineralization

Over the last century, despite enormous advancements in biomedical research and the development of sophisticated analytical instruments many diseases continue to be a burden on humankind particularly on the aged. This is because of a lack of complete understanding of the pathogenesis and specific therapies. Due to the complexity involved, we need to explore all facets of diagnosis and therapies. Therefore, there is a requirement for different strategies to combat these diseases. A quick diagnosis is the primary step towards improving treatment and increasing the chance of survival. To realize this goal we entail to monitor multiple biomarkers which will also help us to understand the progression of disease. Mid-Infrared (MIR) and Raman spectroscopic techniques are well established analytical methods to understand the molecular structure and chemical composition of heterogeneous systems. These techniques are rapid, non-destructive and offer multiple component analysis (global/multiplex) in a single measurement without any labels. Importantly, biological materials like proteins, carbohydrates, lipids, nucleic acids etc. have unique structures and therefore we can obtain unique spectral fingerprints of these molecules in different physiological and pathological conditions. This will provide a potential route to obtain diagnostic markers for diseases. Also, to improve the ability to diagnose and treat human diseases much more efficiently, understanding the mechanisms involved in the progression of disease is necessary. It would be time consuming and often unethical to perform these studies directly on humans. Therefore, there is a need for model organisms to explore the complexity of various diseases. A model organism is an animal, plant or microbe that is being studied to understand a range of biological phenomena. They should meet certain criteria such as short life cycles, easy to breed and maintain in large numbers under laboratory conditions, and the data generated through use of the model should be applicable to other higher organisms like humans. The microbial system, mouse, rat, Drosophila (fruitfly), C Elegans (nematode worm) and zebrafish are being used extensively for this purpose. The most adaptable organisms to study diseases in humans are the mice as they share almost 99% of their genes with humans. Mice are similar to humans in most physiological and pathological features such as nervous, cardiovascular, immune, liver etc. In addition to mice, Drosophila melanogaster (fruitfly) has been used for years as an attractive model organism to understand the mechanisms of underlying human diseases. This is because 75% of human disease genes have counterparts in Drosophila and it meets the above mentioned criteria to be a model organism. It also plays an important role for studying genetics and development biology. The average life span of Drosophila is 60-80 days; therefore it is a suitable model to study age related diseases. In the present thesis, the ability to probe low-micrometer domains using Raman and Fourier Transform Infrared (FTIR) microspectroscopy was utilized to monitor the chemical changes during various biomedical conditions using model systems. Chapter 1 of the thesis discusses about the origin of Raman and FTIR microspectroscopy along with instrumentation and applications. Various data analysis methods (both univariate & multivariate) and the validation criterion are described in chapter 2. Depending on the objective of the study and based on the technique (Raman or FTIR) used, one (or more) of these methods can be applied for effective interpretation of the data. Further, the thesis includes four different investigations; a) the FTIR spectroscopic study of hepatotoxicity due to acetaminophen using mice as model, b) the Raman spectroscopic studies of muscle-related disorders using Drosophila as a model, c) Vibrational spectroscopic study of septic shock using mice as model, d) Surface Enhanced Raman Spectroscopy (SERS) study of serum components using Lab-on-a-chip (LOC). The first part comprises mainly the FTIR microspectroscopy study of hepatotoxicity in mice post oral dosing of acetaminophen (paracetamol), which is extensively used worldwide as an analgesic and antipyretic drug (chapter 3). The infrared spectra of acetaminophen treated livers in BALB/c mice show a decrease in glycogen and an increase in amounts of cholesteryl esters and DNA. Importantly, analysis of sera identified the lowering of glycogen and increase in DNA and chlolesteryl esters earlier than the increase in alanine aminotransferase, which is routinely used to diagnose liver damage. Similar changes are also observed in C57BL/6 and Nos2−/− mice. Revert experiments using an antidote (L-methionine) demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. In the second study Raman spectroscopy is applied to discriminate between various muscle defects in Drosophila, since it can provide a unique molecular fingerprint of tissues on the basis of their biochemical composition (chapter 4). Raman spectra were collected from Indirect Flight Muscles (IFM) of mutants upheld1 (up1), heldup2 (hdp2), Myosin heavy chain7 (Mhc7), Actin88FKM88 (Act88FKM88), upheld101 (up101) and Canton-S (CS) for both 2 and 12-days old flies. The difference spectra (mutant minus CS) of all mutants have shown an increase in nucleic acids (DNA/RNA) content along with an increase in β-sheet and/or random coil content at the expense of α-helix. Interestingly, 12th day sample of up1 & Act88FKM88 exhibit significantly higher levels of glycogen and carotenoids than CS. A Principal Components based Linear Discriminant Analysis (PC-LDA) classification model was developed, which classifies the mutants according to their pathophysiology and yielded overall accuracy (OA) of 97% and 93% for 2 and 12-days old flies respectively. up1 & Act88FKM88 (nemaline myopathy phenotypes) form a group which is clearly separated in a Linear Discriminant (LD) Plane from up101 & hdp2 (cardiomyopathy phenotypes). In the third part we investigated septic shock, a life threatening condition associated with multiple organ dysfunctions, in mice (chapter 5). Salmonella typhimurium were given to BALB/c and 129/SvJ mice via the intraperitoneal route to induce infection. Liver, spleen and sera samples were studied using FTIR microspectroscopy. The infrared spectra of liver, spleen and serum samples in BALB/c (Nramp1-deficient) mice show significant spectral changes as early as 1 hour post infection but spleen shows changes only after 6 hour. Interestingly, 129/SvJ (Nramp1-sufficient) mice were resistant to sepsis and show significant spectral changes only at 12 hour post infection. This study demonstrates that suppression of Nramp-1, a renowned gene known to control susceptibility to infections by intracellular bacteria can be an effective cure for sepsis. The final study presented in this thesis demonstrates the use and benefits of lab-on-a-chip (LOC) devices in surface enhanced Raman spectroscopy (SERS) which is used to enhance the weak Raman signals (chapter 6). Most of the diseases have related proteins or analytes present in serum although in early stages their concentration in blood are low. The idea is to detect at low concentration using SERS the serum components which are related to progression of disease. Here, we have compared the effect of different aggregating agents on silver colloids and the resulting enhancement in Raman signals for tryptophan and Bovine Serum Albumin (BSA). Reproducibility issues, the key concern of static phase SERS, can be overcome by performing SERS spectroscopic measurements in automated flow cells. Further, pyridine and tryptophan were used to demonstrate SERS in a segmented flow system. The spectra from different drops were compared and demonstrate the high reproducibility in comparision to static SERS. Lastly, chapter 7 summarizes the entire work of the present thesis with future prospects of Raman and FTIR microspectroscopy to study the progression mechanism of various diseases like neurodegenerative diseases which is easy to follow in drosophila due to their short life span. Also, technological developments in the field of nanotechnology and micro-fluidics will enable the detection of early biochemical changes in bodily fluids such as urine, cerebral spinal fluid, tears etc. Building on the results demonstrated in this thesis, hopefully label-free vibrational (Raman and FTIR) microspectroscopic studies using model organisms would help in understanding the underlying mechanisms of progression of various other diseases which in turn would facilitate the development of effective therapies.

Our work falls within the field of high resolution spectroscopy of gas phase molecules in the far-, mid- and near-infrared ranges. Its guiding line are absorption spectral intensities, dealt with experimentally and theoretically. In particular, we developed in our laboratory the field of intensities measurements of vibration-rotation lines using Fourier transform spectrometers, with a precision of about 0.5 % and an accuracy of 2-4 % for chemically stable species. We study chemically stable (CO2, N2O, C2H6, OCS, C2H2) and unstable (HOCl, HCOOH et HNO3) compounds. We also measure infrared absorption cross section spectra for CFC replacements. Some of our measured intensities, of direct interest for the study of planetary atmospheres, are now incorporated in international spectroscopic databases. Others are used to develop global theoretical models of molecules, in relationship with intra-molecular dynamics./Nos travaux de recherches relèvent de la spectroscopie à haute résolution de molécules en phase gazeuse dans les domaines de l’infrarouge lointain, moyen et proche. Ils ont pour ligne directrice les intensités spectrales d’absorption, abordées expérimentalement et théoriquement. Nous avons en particulier développé dans notre laboratoire le domaine de la mesure d’intensités d’absorption de raies de vibration-rotation à l’aide de spectromètres à transformée de Fourier. Nous réalisons ces mesures avec une précision d’environ 0.5 % et une exactitude d'environ 2-4 % pour les espèces chimiquement stables. Nous étudions des composés chimiquement stables (CO2, N2O, C2H6, OCS, C2H2) et instables (HOCl, HCOOH et HNO3). Nous mesurons également des spectres de sections efficaces d’absorption infrarouge pour des substituts des chlorofluorocarbures (CFC). Certaines de nos mesures d’intensité, d’intérêt direct pour l’étude d’atmosphères planétaires, sont aujourd’hui incorporées dans les bases de données spectroscopiques internationales. D’autres sont utilisées pour développer des modèles théoriques globaux de molécules, en liaison avec la dynamique intramoléculaire. / Agrégation de l'enseignement supérieur, Orientation sciences / info:eu-repo/semantics/nonPublished