Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression. / February 2008

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression.

Infrared microspectroscopy has been used to survey Alzheimer’s diseased brain tissue from a transgenic mouse model of the disease. Alzheimer’s disease is the leading cause of dementia among the elderly and is characterized by β-amyloid plaque deposition,neurofibrillary tangles, inflammation, and disturbed energy metabolism in the brain. Both the TgCRND8 and Tg19959 mouse models of the disease develop Alzheimer’s disease pathology beginning at approximately 3 months of age. Infrared microspectroscopy allows analysis of untreated, flash frozen tissue samples, at micron level spatial resolution, and was used in this study to examine creatine deposits in the Alzheimer’s diseased brain. Creatine is central to cellular energetics and plays an important role in proper brain function. The hippocampi of 7 pairs of transgenic mice and their littermate controls were mapped using infrared microspectroscopy and the results were analyzed for creatine levels and levels of β-sheet, indicative of the presence of β-amyloid plaques. Creatine was found to be focally elevated in the transgenic mice, as compared to their littermate controls but was not co-localized with β-amyloid plaques. Further surveys of serial sections from one transgenic mouse showed the 3-dimensional distribution of creatine within the sample. Focally elevated creatine may be a marker of the disease process, indicative of disturbed energy metabolism or inflammatory response to the disease progression.

Dry matter, crude protein and starch degradation characteristics of one corn (Pioneer 39P78) and four barley grain varieties (CDC Bold, CDC Dolly, Harrington and Valier) were evaluated in two in situ nylon bag trials. Trial 1 compared ground and rolled treatments of Harrington barley and Pioneer 39P78 corn, whereas Trial 2 evaluated ground and rolled treatments of the four barley varieties. Rumen degradability characteristics were compared with analytical results from thermal- and synchrotron-source FTIRM. Infrared absorbance spectra were collected from corn and four barley varieties using thermal-source FTIRM on the mid-IR beamline at the Canadian Light Source, Ltd. (Saskatoon, SK). Synchrotron-source FTIRM spectral data was collected for corn, Harrington barley and Valier barley on the U2B mid-IR beamline at NSLS-BNL (Upton, NY). CHO:Amide I peak area ratios were compared to the in situ rumen degradation results to determine if FTIRM spectral data could be related to the rate and extent of rumen degradation, and if thermal- and synchrotron-source FTIRM yielded different results. A grain x processing method interaction (P<0.01) was observed in both in situ trials where grinding produced a greater increase in the rate and extent of rumen degradation for Harrington barley than it did for corn (Trial 1) along with a greater increase in the rate and extent of rumen degradation for CDC Bold and CDC Dolly than for Harrington and Valier (Trial 2). Among barley varieties, increasing rate and extent of rumen degradation (CDC Bold>CDC Dolly>Harrington>Valier) corresponded to increasing starch:protein ratio as estimated by chemical analysis. This relationship was reversed for corn and Harrington barley where corn had a higher starch:protein ratio yet slower rumen degradation kinetics. For both thermal- and synchrotron-source FTIRM, CHO:amide I peak area ratios were greater (P<0.05) for corn than for Harrington barley. Comparison of CHO:amide I peak area ratios of barley varieties measured with thermal-source FTIR showed that varieties with higher (P<0.05) CHO:Amide I peak area ratios generally had higher rate and extent of rumen degradation. This indicates that starch:protein ratio estimated with FTIRM may be an indicator of rumen degradability characteristics when comparing varieties of the same grain, but not for different species of grains.

Dry matter, crude protein and starch degradation characteristics of one corn (Pioneer 39P78) and four barley grain varieties (CDC Bold, CDC Dolly, Harrington and Valier) were evaluated in two in situ nylon bag trials. Trial 1 compared ground and rolled treatments of Harrington barley and Pioneer 39P78 corn, whereas Trial 2 evaluated ground and rolled treatments of the four barley varieties. Rumen degradability characteristics were compared with analytical results from thermal- and synchrotron-source FTIRM. Infrared absorbance spectra were collected from corn and four barley varieties using thermal-source FTIRM on the mid-IR beamline at the Canadian Light Source, Ltd. (Saskatoon, SK). Synchrotron-source FTIRM spectral data was collected for corn, Harrington barley and Valier barley on the U2B mid-IR beamline at NSLS-BNL (Upton, NY). CHO:Amide I peak area ratios were compared to the in situ rumen degradation results to determine if FTIRM spectral data could be related to the rate and extent of rumen degradation, and if thermal- and synchrotron-source FTIRM yielded different results. A grain x processing method interaction (P<0.01) was observed in both in situ trials where grinding produced a greater increase in the rate and extent of rumen degradation for Harrington barley than it did for corn (Trial 1) along with a greater increase in the rate and extent of rumen degradation for CDC Bold and CDC Dolly than for Harrington and Valier (Trial 2). Among barley varieties, increasing rate and extent of rumen degradation (CDC Bold>CDC Dolly>Harrington>Valier) corresponded to increasing starch:protein ratio as estimated by chemical analysis. This relationship was reversed for corn and Harrington barley where corn had a higher starch:protein ratio yet slower rumen degradation kinetics. For both thermal- and synchrotron-source FTIRM, CHO:amide I peak area ratios were greater (P<0.05) for corn than for Harrington barley. Comparison of CHO:amide I peak area ratios of barley varieties measured with thermal-source FTIR showed that varieties with higher (P<0.05) CHO:Amide I peak area ratios generally had higher rate and extent of rumen degradation. This indicates that starch:protein ratio estimated with FTIRM may be an indicator of rumen degradability characteristics when comparing varieties of the same grain, but not for different species of grains.

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.

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

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

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / The fate and bioavailability of organic contaminants in plants is a major ecological and human health concern. Current wet chemistry techniques that employ strong chemical treatments and extractions with volatile solvents, such as GC-MS, HPLC, and radiolabeling, although helpful, degrade plant tissue resulting in the loss of spatial distribution and the production of artifacts. Synchrotron radiation infrared microspectroscopy (SR-IMS) permits direct analysis of plant cell wall architecture at the cellular level in situ, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers such as cellulose, lignin, and proteins in the root tissue of hydroponically grown sunflower and maize plants as well as to determine the fate and effect of several organic contaminants in those root tissues. Principal components analysis (PCA), a data compression technique, was employed to reveal the major spectral variances between untreated and organic contaminant treated root tissues. Treatment with 1H-benzotriazole (BT) caused alterations to the lignin component in the root tissue of plants. The BT was found in xylem and epidermal tissue of sunflower plants but not associated with any particular tissue in maize roots. 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) altered the pectin and polysaccharide structure in both maize and sunflower. SR-IMS revealed the reduction of DNTs to their aromatic amine form in the vascular and epidermal tissues at low concentration. At high concentration, DNTs appeared to be associated with all the plant tissues in maize and sunflower. Exposure of sunflower and maize to 2,6-dichlorophenol (2,6-DCP) caused alterations to the polysaccharide and protein component of the root tissue. In some cases, phenolic compounds were observed in the epidermal tissue of maize and sunflower roots. The results of this research indicate that SR-IMS has the potential to become an important analytical tool for determining the fate and effect of organic contaminants in plants.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / During germination, biochemical changes occur in the wheat kernel by stimulation of enzymes and hormones, and the seed reserves are mobilized. Infrared microspectroscopy and imaging enables a localized chemical inventory, upon germination, to study the process. Frozen sections of germinated wheat mounted onto BaF[subscript]2 were mapped to produce functional group images for comparison with corresponding sections of ungerminated kernels. Relative functional group populations in the scutellum and embryonic axis were assessed before and after germination. An average 23% reduction in lipid to protein ratio was observed in the scutellum based on the comparison of 53,733 spectra. As a result of the early germination process, lipid in the scutellum was depleted to provide energy for the growing embryo. Germination of the kernels while in the field before harvest due to high humidity is known as preharvest sprouting. Preharvest sprouting has detrimental effects on the end use quality of the wheat (sprout damage) and cause economic loses. Tolerance to preharvest sprouting is highly desirable. To assist breeding program, a nondestructive near-IR chemical imaging method has been developed to test new lines for resistance to preharvest sprouting. The higher sensitivity of subsurface chemical imaging, compared with visual detection, alpha amylase determination, or viscosity testing, permits germination detection at early stages. A near-IR chemical imaging system with an InGaAs focal plane array (FPA) detector in the 1100 nm-1700 nm range was used. Kernels from six different cultivars, including HRW and HWW wheat, were exposed to moist conditions for 6, 12, 24, 36, and 48 hours. Images of each 90 kernel group were examined; kernels exposed to moisture for 36 hours were compared with kernels treated for 3 hours as a control. Each kernel was classified as sprouted or not sprouted with the criteria of log 1/R intensity at select wavelengths or select factors of principle component analysis (PCA) treatment of reflectance intensity data. Imaging wavelength range was expanded beyond 1700 nm to 2400 nm with the use of InSb FPA. Study for the potential for unsupervised determination in nondestructive near-IR imaging with detection wavelengths 1200-2400 is ongoing. Some preliminary results presented are encouraging.