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Near infrared (NIR) spectroscopy is being developed on living tissue models for noninvasively measuring in vivo glucose concentrations in individuals with diabetes. Multivariate calibration models have been built and the selectivity of each multivariate signature has been evaluated by several means. The primary objective of the research detailed in this dissertation is to practically apply noninvasive NIR glucose measurements on animal models for both short-term and long-term studies and preview future human subject evaluations. In the animal study, living tissue spectra were collected through a modified optical interface with hyper- and hypo-glycemia control. Selective measurements of glucose molecules are illustrated by the partial lease squares (PLS) algorithm, net analyte signal (NAS) vector, and hybrid linear analysis (HLA). Each model demonstrates the ability to predict prospective glucose concentrations in the short term. A restraint platform was developed for the long-term study on conscious animals. Conscious animal spectra were collected on multiple days. The anesthetized animal experiment follows on the final day. Principal component analysis (PCA) of spectra collected on different days demonstrates no significant difference between conscious animal spectra and anesthetized animal spectra. Moreover, an NAS vector analysis from conscious animal spectra has the ability predict glucose concentrations which follow the blood glucose transient during the anesthetized animal experiment. This procedure has great potential to be applied in future NIR glucose monitoring device. Before the application of this noninvasive NIR technology on people with diabetes, the impact of skin difference must be determined. In this human subject study, human skin color and baseline spectra were collected and analyzed to determine differences among individuals and within groups of people. To compare in vivo NIR spectra with different skin characteristics, PCA was performed to obtain principal component (PC) scores. Poor correlation between PC scores and skin characteristics concludes that noninvasive near-infrared technology is insensitive to different types of skin. In addition, glucose prediction was performed by a NAS analysis. The prediction results demonstrate that it is feasible to build a NAS glucose model for noninvasive NIR glucose predictions in human subjects.

Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

Carisbamate (CRS) and lamotrigine (LTG) are anticonvulsants which act mainly on neuronal voltage-gated sodium channels, that have been shown to have antidepressant-like effects in animal models of depression. In vivo electrophysiological recordings were carried out following 2 and 14 days of CRS or LTG administration. Overall firing activity in the dorsal raphe, locus coeruleus and ventral tegmental area were decreased with CRS. Similarly, a decrease in the dorsal raphe was also observed with LTG. Despite these presynaptic decreases in firing activity, both anticonvulsants exhibited significant enhancement of serotonergic transmission in the hippocampus as demonstrated by increased tonic activation of postsynaptic 5-HT1A receptors. This may be attributed to the observed desensitization of the terminal 5-HT1B autoreceptors. This study suggests that the enhanced serotonergic effect may be associated with an antiglutamatergic effect, and may contribute to the antidepressant-like effect of CRS in the forced swim test and the antidepressant properties of LTG.

An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented. A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light. The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils. Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation. A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated. Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.

An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented. A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light. The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils. Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation. A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated. Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.

The presence of off-flavor compounds in fish represents one of the significant economic problems encountered in aquaculture. These off-flavor compounds are due to the absorption of substances produced by microorganisms. Currently, a number of strategies have been employed to prevent or limit the growth of these microorganisms in recirculating aquaculture system. Therefore, it is important to evaluate the effectiveness of these strategies by monitoring the concentrations of off-flavor compounds in fish. In-vivo solid phase microextraction (SPME), a rapid and simple sample preparation method, allows the monitoring of concentrations of off-flavor compounds in live fish. In this research, geosmin and 2-methylisoborneol (2-MIB), which are produced by cyanobacteria and actinomycetes being the major sources for “earthy” and “muddy” flavors in fish, were selected as representatives. In order to accurately quantify these compounds in fish muscle, two kinetic calibration methods, on-fibre standardization and measurement using pre-determined extraction rate, were used. Results obtained were validated by traditional methods. The detection limit of in-vivo SPME in fish muscle was 0.12 ng/g for geosmin and 0.21 ng/g for 2-MIB, both below the human sensory threshold. Additionally, the binding effect of geosmin and 2-MIB in fish muscle was investigated in details. Facilitated by the agarose gel model, it was proven that binding did not impact the extraction rate under the pre-determined sampling time. Furthermore, an optional sampling position was undertaken by inserting the fibre into the fat tissue found under the fish belly, the results indicating that this method could decrease extraction time by up to two-thirds of its usual time.

To evaluate the anticancer potential of sorghum phenolic compounds, different experiments including in vitro and in vivo tests were performed. A set of 25 sorghum samples was evaluated for phenolic (total phenols, flavonoids, anthocyanins and tannins) content, hydrophilic and lipophilic antioxidant capacity using de Oxygen Radical Absorbance Capacity assay (ORAC), and screened for citotoxic properties in mammary, colon and hepatic mammalian cancer cell lines in vitro. Results indicated that there was a wide variability in the phytochemical profile among the different sorghums. Among the 25 samples, sumac sorghum bran had the highest amount of phenolic compounds, flavonoids, tannins and the highest ORAC values. It exerted the highest percent inhibition (near 100%) in mammary, colon and liver cancer cell lines. Sumac sorghum bran was selected for further investigation. Methanolic extracts from sumac whole grain, bran and tannin removed bran were tested in vitro at different concentrations in hormone dependent MCF-7 mammary cancer cells and non hormone dependent Caco2 and HepG2 colon and liver cancer cells. Results indicated that the methanolic extract from sumac bran inhibited 100% of MCF-7 cancer cells at a concentration of 0.5 mg/ml and that the citotoxic effect could be partially due to the tannin content of the extract. Concentrations of 0.5 and 1.5 mg/ml were selected for an in vivo preventive cancer study with 7,12-dymethylbenz(a)-anthracene (DMBA) induced female rats. Bran at low and high concentrations and the correspondent amount of methanol extracts were included in the diet. It was observed that sumac methanol extract at low concentration promoted tumor appearance and development, whereas sumac bran had a preventive effect, however, there were no significant differences in rats treated and un-treated with sumac. Differences between in vitro and in vivo results could be due to the degree of absorption of tannins during the in vivo experiment. To obtain additional data about the effect of sumac extracts on cancer development, a quinone reductase enzyme bioassay was performed. Methanol and hexane extracts from sumac bran induced phase II enzymes in vitro. Phytochemicals of sumac bran sorghum including phenolic compounds and lipid like compounds appeared to have potential for cancer prevention.