Vasomotion is the spontaneous oscillation of vascular tone, occurring due to synchronization of internal calcium fluctuations between multiple vascular smooth muscle cells by gap junction and electrical communication. Although altered vasomotion has been observed in a variety of pathological situations, characterization of these alterations has been lacking. Using a novel method of spectral quantification, and two experimental models known to have altered vascular structure, the present thesis was designed to evaluate whether vasomotion characteristics could be correlated with altered vascular structure. Rats with perinatal iron deficiency (PID) have previously been shown to possess altered vascular structure. When phenylephrine-mediated contractile and acetylcholine-mediated dilatory responses were investigated in PID animals, they both displayed blunted relaxation as compared to control vessels. When vasomotion characteristics were quantified, vessels taken from PID animals exhibited a decreased power in the very low frequency window (VLF <0.2 Hz). Changing vessel oxygenation to 10% O2 from 95% O2 did not result in significant alterations of vasomotion characteristics. The primary frequency of oscillation was investigated with a peak finder, and found to be significantly different compared to control in both the aorta and renal arteries obtained from PID animals. To investigate the effect of antihypertensive treatment (enalapril and hydrochlorothiazide) on gap junction communication, spontaneously hypertensive rats (SHR) were subject to a 2-week intensive angiotensin converting enzyme inhibitor treatment. This treatment resulted in significant vascular structural regression. All vessels (aorta, renal, mesenteric) from treated animals had greater proportions of power in the VLF window, with both the mesenteric and renal vessels exhibiting a primary peak of oscillation around 0.2 Hz; whereas the aorta had a primary peak at 0.12 Hz. Investigating altered gap junction communication with the gap junction blocker 18-α glycyrrhetinic acid, revealed that vascular bed location was the determining factor of vasomotion response. Immunoblotting did not indicate differences in connexin 43, a major gap junction protein in the vascular smooth muscle. These studies suggest that vasomotion characteristics can be used as a method of vascular phenotype investigation; vasomotion characteristics were significantly different in vessels taken from PID and hypertensive animals as compared to control and antihypertensive-treated animals, respectively. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2008-09-26 11:39:44.043

Three new quantitative Fourier transform infrared (FTIR) spectroscopic methods were developed for the analysis of edible oils: two procedures to measure free fatty acids (FFA) and one to measure moisture (H2O), the latter two methods ultimately being automated and implemented on an auto-sampler equipped FTIR spectrometer. The methods developed for FFA determination both convert FFAs to their carboxylate salts by means of acid/base reaction without causing oil saponification, one approach using 1-propanol, an oil-miscible solvent, and the other using methanol, an oil-immiscible solvent into which the FFA salts are extracted. The first method involves splitting oil samples into two halves, with one half treated with propanol containing base and the other half with propanol only. The spectra of each half is collected and a differential spectrum obtained, from which quantization is performed. The methanol procedure simply involves extracting FFA into methanol containing a weak base and quantitating the FFA salts produced. Both FFA methods determine the FFA content by measuring the v (COO-) absorbance at &sim;1570 cm-1 relative to a reference wavelength of 1820 cm-1 from a differential spectrum relative to the solvent, the extraction procedure being superior in terms of both speed and sensitivity, being able to measure FFA levels down to &sim;0.001%. The method developed for moisture determination involves extracting water in edible oils into dry acetonitrile and then quantitating it by measuring the absorbance of the OH stretching band (3629 cm-1) and/or the HOH bending band (1631 cm -1). All three methods were validated by standard addition experiments, evaluated for potential interferences, and, in the case of FFA determination, compared to the performance of AOCS official methods. The results indicated that the extraction-based procedures were superior to conventional wet chemical methods in both sensitivity and reproducibility. The FFA and H2O extraction procedures were subsequently automated by connecting an auto-sampler to the FTIR spectrometer and developing procedures and software algorithms to enable the analysis of up to 100 samples/h. The methods developed and implemented are a substantive improvement over conventional methods for the analysis of FFA and H2O in edible oils and provide a means by which QC and process laboratories can analyze large volumes of edible oils for these two important parameters.

A mid-infrared chalcogenide fiber optic probe was employed to develop a Fourier transform infrared spectroscopy-based partial-least-squares (PLS) calibration model for the quantitative analysis of sucrose, glucose, fructose, maltose, total sugar and water content in chocolate syrup. Based on the comparison of the pure component and correlation spectra extracted from chocolate syrup and aqueous sugar solutions based models, it was determined that the tightness of the concentration ranges and the ratios of the sugars in the chocolate syrup samples did not allow to draw adequate information to build a robust PLS calibration model. PLS regression models developed using infrared spectra of chocolate syrup calibration standards prepared by addition of sugar solutions to increase the concentration range did not yield conclusive results. A different approach used for standard preparation consisted of diluting chocolate syrup samples to different degrees. This new method provided an increased concentration range for the sugars but maintained an almost constant sugar to sugar ratios. The PLS models based on these new calibration standards yielded high calibration correlation coefficients and low errors on the external validation. Accuracy, repeatability, long-term stability and ruggedness were tested and the results demonstrated that the calibration models were robust and had a better repeatability than the reference high-performance liquid chromatography method. The fact that the calibration model was developed using standards having very similar sugar profiles precluded its use for the analysis of chocolate syrup samples of different formulations. The resulting formulation-specific PLS regression model required a preclassification step to ensure that the model is applied to the appropriate sample type. A probabilistic neural network (PNN) model was developed to fulfill the preclassification requirement. PNN yielded excellent classification results. The modeling uncovered

The application of infrared imaging employing focal plane array Fourier transform infrared (FPA-FTIR) instrumentation for the identification of bacteria was investigated. FPA-FTIR spectroscopy was shown to provide new opportunities for bacteria identification with unprecedented reliability and throughput by allowing 102--103 FTIR spectra to be acquired simultaneously from surface areas of 90 x 90 to 200 x 200 mum with a spatial resolution of &sim;6 mum. The combination of data redundancy and spatial resolution afforded by infrared imaging made it possible to acquire highly reproducible spectra from bacterial films. A protocol for enhancing the reliability of bacteria identification by transmission-mode FPA-FTIR spectroscopy was developed by optimizing spectral acquisition parameters, spectral processing and data analysis; using the differentiation of two Campylobacter species as a test case. The results for this test case were compared with those obtained from three alternate FTIR spectral acquisition modes. The optimized protocol was employed for the generation of a spectral database of foodborne bacteria, containing over 1,000,000 spectra acquired by infrared imaging of 36 species from 19 genera. The development of a modular hierarchical clustering (MHC) model, in combination with the use of a region selection algorithm, allowed all species in the database to be differentiated from each other down to the species level based on differences in their infrared absorption profiles. A validation study involving the identification of well-characterized isolates by comparison of their spectra to those in the database demonstrated the robustness of the MHC model. In a further study employing 44 strains of Clostridium botulinum, the discriminatory power of FPA-FTIR spectroscopy was compared with that of pulsed-field gel electrophoresis, and the region selection algorithm was applied to identify growth medium-independent spectral regions that allowed for the differentiation of Group I and Group II C. botulinum strains in two blind validation studies. The research carried out also demonstrated the high-throughput potential of bacteria identification by infrared imaging when combined with the use of a microarray system for sample deposition. Overall, the novel FPA-FTIR spectroscopy-based bacteria identification protocol developed in this work provides a rapid-response and reagent-free technique suitable for routine use in both food and clinical microbiology laboratories.

Globular proteins are important ingredients in many food formulations because of their nutritional value and their various functional properties such as gel formation. Proteins form gels by polymerization into a three-dimensional matrix. The rheological properties of the resulting viscoelastic solids can be obtained by Instron measurements. In the present work, gels were obtained by heating solutions of bovine serum albumin (BSA) in D$ sb2$O and egg albumin in H$ sb2$O under different conditions (i.e., pH, salt concentration, protein concentration, time of heating and temperature), and their gel strengths were measured by a compression test (Universal Testing Machine LRX). The Fourier transform infrared (FTIR) spectra of the same gel samples were recorded in order to investigate the changes in protein structure at the molecular level accompanying gel formation. Intermolecular $ beta$-sheet formation was found to increase as gel formation progresses at the expense of intramolecular $ beta$-sheet and $ alpha$-helix structures. For BSA, maximum gel strength was obtained around pH 7. The addition of salt had little effect on the gel strength while increase in protein concentration, time of heating and temperature increased the gel strength. The rate of denaturation of BSA and ovalbumin and of mixtures of these proteins in the ratios 9:1, 1:1, and 1:9 was also investigated by measuring the peak height of an aggregation band at 1618 cm$ sp{-1}$; some inhibitory effects of BSA on ovalbumin aggregation were observed. Correlations between the measured gel strengths and the amide I band profile in the FTIR spectra were examined using partial-least-squares (PLS) regression. These studies reveal that gel strengths of a particular protein gel could be adequately predicted from their infrared spectra without the need to carry out the rheological compression measurements.

This work describes an investigation of the development of a Fourier Transform Infrared (FTIR) spectroscopic method for the determination of Solid Fat Index (SFI) of fats as a possible replacement of the traditional dilatometric procedure. The initial approach considered was the use of an existing FTIR analytical package which was designed to measure iodine value (IV), saponification number (SN), and cis and trans content. It was hypothesized that these measures could be related to SFI using multiple linear regression (MLR), thereby allowing the existing analytical package to simultaneously make SFI measures. It was found that there was a strong relationship between SN/cis/trans measurements and SFI, especially in sequentially hydrogenated oils. The MLR relationships, however, did not reproduce the dilatometric SFI values with sufficient accuracy in the general case, and this approach had to be abandoned. Subsequently, a partial least squares (PLS) calibration approach was investigated, relating the dilatometric SFI data directly to the spectral characteristics of the melted fats. It was found that suitable PLS calibrations could be developed for soybean and Canola oils. Based on these results, an FTIR system was programmed to determine SFI and the performance of the system validated using pre-analyzed "unknowns". It was shown that the SFI of either soybean or Canola oils could be determined to within $ pm$ 1.0 SFI. As configured, the FTIR system is capable of determining the SFI of a neat and clear, melted fat sample at 80$ sp circ$C in less than two minutes, providing four SFI values, representing the solids content at 50, 70, 80 and 92$ sp circ$F. In contrast to the standard dilatometric method, which takes over two hours to carry out, the FTIR approach provides a rapid means of determining SFI, the technique being suitable for routine quality control applications in the fats and oils industry.

Staphylococci strains are among the most widespread multidrug-resistant nosocomial pathogens in Canada. Rapid and accurate identification and epidemiological typing of methicillin-resistant S. aureus (MRSA) and its discrimination from coagulase-negative staphylococci (CNS) and glycopeptide-intermediate S. aureus (GISA) are crucial for appropriate therapy and for monitoring and limiting intra- and inter-hospital spread of epidemic MRSA strains. Although pulsed-field gel electrophoresis and polymerase chain reaction methods for the identification of MRSA are reliable, they are technically demanding, time-consuming and inappropriate for routine clinical diagnosis. Moreover, no reliable method exists for discrimination of epidemic MRSA from sporadic MRSA and from GISA strains. The objective of the research described in this thesis was to investigate whether Fourier transform infrared (FTIR) spectroscopy could be used to distinguish MRSA from methicillin-susceptible S. aureus, borderline oxacillin-resistant S. aureus (BORSA), CNS, including methicillin-resistant CNS, and GISA. The application of FTIR spectroscopy for epidemiological typing of Canadian epidemic MRSA (CMRSA) strains as well as their discrimination from sporadic MRSA was also assessed. FTIR spectra were recorded from intact stationary-phase cells grown on Universal Medium (UM™) and deposited and dried on a ZnSe optical window, normalized, and converted to first-derivative spectra. Various chemometric approaches were employed to cluster the different phenotypes of staphylococci species and to subtype five CMRSA strains based on the similarity of their infrared spectral fingerprints in narrow spectral regions selected by visual inspection and by employing a singularvalue decomposition (SVD) algorithm. Pairwise separation of MRSA from MSSA, BORSA, CNS, MRCNS, and GISA was accomplished by using principal component analysis (PCA), self-organizing maps (SOM), and the K-nearest neighbors (KNN) algorithm. These chemometric techniques were also successfully employed for epidemiological typing of the five CMRSA strains and their discrimination from sporadic MRSA strains using a combination of different optimal spectral regions selected by SVD. These results demonstrate that FTIR spectroscopy has considerable potential as a rapid method for the identification of different phenotypes of staphylococci and epidemiological typing of MRSA.

Variable-temperature Fourier transform infrared spectroscopy can be employed to monitor the denaturation and aggregation of proteins during heat treatment. Information on the changes that occur in protein secondary structure upon heating is provided by detailed examination of the amide I band, as different protein conformations have characteristic amide I frequencies. The objectives of the present study were: (i) to study the changes in protein structure that occur during gelation of whey protein isolate (WPI) and (ii) to correlate the changes in protein structure observed under different physico-chemical conditions to rheological properties of WPI gels prepared under the same conditions. / The FTIR spectra of D2O solutions of WPI at different pHs, ranging from 3 to 10, were recorded as the temperature of the solution was increased from room temperature to 95°C in 5°C increments. In all the solutions studied, the formation of two new bands at 1618--1623 cm -1 and 1678--1684 cm-1 was observed upon heating; these bands are characteristic of aggregate formation and have been previously assigned to antiparallel beta-sheet structure. As the pH increased from 3 to 10, the aggregation temperature of WPI decreased from 85°C to 65°C. / The rheological properties of WPI gels were studied by employing an Instron Universal Testing Machine. The Instron measurements demonstrated that protein concentration, heating temperature, and heating and cooling time are directly related to gel strength. The changes in gel strength as a function of cooling time (for gels prepared by heating at 75°C for 45 min) were correlated to FTIR spectral data for WPI solutions subjected to the same treatment.

Recent studies have demonstrated that applied hydrostatic pressure can affect the functional properties of whey protein isolate (WPI). In this work, the effects of applied hydrostatic pressure on the tertiary and secondary structure of whey proteins were investigated by spectroscopic and rheological techniques to elucidate the molecular basis of such pressure-induced changes in protein functionality. The individual protein components of WPI and various samples of WPI obtained from different sources were subjected to different single-cycle pressure treatments of up to 400 MPa in 100 MPa increments with 30-min holding time as well as to pressures ranging from 450 to 650 MPa without a holding time. Electrospray ionization-mass spectrometry, circular dichroism, and Fourier transform Raman spectroscopic studies of pressure-treated samples of beta-lactoglobulin, the major protein component of WPI, revealed significant changes in tertiary structure. Fourier transform infrared spectroscopic studies revealed that the secondary structure of beta-lactoglobulin was also sensitive to applied pressure and holding time. The secondary and tertiary structure of alpha-lactalbumin, the second most prevalent protein in WPI, was unaffected by applied hydrostatic pressure. The spectroscopic behaviour of the various samples of WPI subjected to pressure treatment was variable and indicated that the response of WPI to applied hydrostatic pressure is dependent on the method used to isolate the WPI from whey. The rheological profiles of beta-lactoglobulin, alpha-lactalbumin, and WPI samples after various pressure treatments were also recorded. Both beta-lactoglobulin and WPI exhibited marked increases in viscosity with increasing pressure, whereas alpha-lactalbumin remained solutions exhibited no significant change in viscosity. These studies have furthered the understanding of the effects of applied hydrostatic pressure on the molecular structure and rheological pr

Fourier transform infrared (FTIR) spectroscopy is a powerful tool for probing protein structure-function relationships. With the use of isotope editing, it can also be employed to elucidate protein-nucleic acid interactions. This technique was used to study the sequence of heat-induced unfolding of the uniformly labeled 13C regulatory subunit (RSU) of E. coli aspartate transcarbamylase (ATCase) with its inhibitor CTP. The absorption of CTP in the amide I' region limits our ability to detect protein conformational changes upon binding of CTP. Therefore, by labeling the protein with 13C shifts the amide I' band ~ 40 cm -1 and clearly separates the protein bands from those of CTP. Variable-temperature (VT) FTIR spectroscopy was then employed to monitor the thermal unfolding of the labeled RSU in the presence and absence of CTP. / In addition, isotope editing was further explored to probe domain-domain interactions of the two domains of RSU using intein technology. Intein technology provides a novel means by which isotope editing can be performed to extract information on protein inter-domain and inter-subunit interactions by spectroscopic analysis but has not yet been exploited in Fourier transform infrared (FTIR) spectroscopy. The objective of this project is to present for the first time the feasibility of segmental labeling through intein-mediated protein ligation (IPL) for the purpose of studying conformational changes by FTIR spectroscopy, using ATCase as a model enzyme. In the first phase of this project, the RSU of ATCase, which houses a Zn-binding domain and a nucleotide binding domain, was reconstructed from its isolated domains using commercially available intein-base expression vectors. As steps towards obtaining an isotope labeled RSU, we have fused each domain to separate inteins. Following affinity purification, the intein tags were chemically cleaved and the reactive ends of the two RSU domains were ligated together to form a peptide. Although ligation was successful, improved yields are required for the FTIR spectroscopic studies.