Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea). This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions. Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst. The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp. Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). <br /><br /> Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules. <br /><br /> Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

In this thesis, a novel electrochemical array is reported. The array consists of two planar halves, each having four carbon screen-printed band electrodes (SPEs), orthogonally facing each other and separated by a spacer to yield 16 two-electrode electrochemical cells with 1 mm² working electrode areas. The 16 counter electrodes were converted to Ag/AgCl by electrodeposition and anodization. These electrodes were stable for at least 30 days with potentials under the current densities used in our experiments. The 16 working electrodes were modified by Au electrodeposition, and were examined by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). <br /><br /> Immobilization strategies for biomolecules are of paramount importance for successful fabrication of biosensors. This thesis reports a new immobilization method that is based on patterned deposition of alkyl thiosulfates (Bunte salts). Monolayers were formed through electrochemical oxidation of Bunte salts at Au-modified electrodes. Single-component and mixed monolayers were investigated, where the mixed monolayers involved one component with a terminal carboxylic acid functional group to allow immobilization of biomolecules. <br /><br /> Applications of the newly developed immobilization method to an enzyme-based biosensor and an immunosensor were investigated. Glucose and biotin were chosen as model analytes, respectively. Glucose oxidase (GOx) and avidin were covalently immobilized onto the mixed-monolayer-modified electrodes through the carboxylic acid groups. Under the optimized conditions for the fabrication and operation of the biosensors, the new electrochemical array showed linearity up to 10 mM glucose with a sensitivity of 4. 7 nA mM^-1 and a detection limit of 0. 8 mM (S/N=3), and linearity up to 12. 8 µM biotin with a detection limit of 0. 08 µM (S/N=3).

Mobility of the horse to initiate motion from the standing position is examined in this thesis. In particular, the thesis focuses on the study of the mobility of a horse with fixed hooves to the ground, and on how its musculoskeletal system is used to free the legs from restraints. Possible leg patterns to initiate motions are investigated. The breaking forces generated at front and hind hooves during static-pulling and dynamic jerking are evaluated. Design of the restraint system that uses ropes to immobilize certain joints in order to prevent the horse from generating these forces is the main objective of this thesis. Such a system could be applied as an alternative to rather massive mechanical devices, the main purpose of which is to block the breaking forces (which are quite large when fully developed).<p> Analysis of the mobility of the horse is based on the mechanics of a skeletal linkage system driven by muscle forces. Only major muscles involved in fighting the restraints are included in the analysis. The force generation capability of a muscle is determined by physiological cross sectional area (PCSA) of the muscle. Possible leg patterns are predicted with the kinematics analysis considering range of motion at each joint in the legs. Corresponding breaking forces generated in each pattern is evaluated with the kinetics analysis. Relationship between the characteristic parameter of the pattern and the breaking force at hoof are established. <p>The horse's computer model is used to justify the analytical result. Fighting mechanisms of the horse are simulated in the dynamic simulation software package. Patterns and the breaking forces developed by the horse model simulation agree well with the analytical results. To the authors best knowledge, this is the first time a computer model is used in analyzing the method of restraining an animal. <p>The mobility of the animal with hoof restraints and methods to remove mobility were further confirmed with a preliminary animal restraint test conducted on a sheep. The sheep was chosen because the leg patterns to initiate motion on a horse are similar to that of sheep, but the sheep is more convenient to handle. The experiment showed that the mobility of the sheep could be removed completely by restraining its hooves, lower legs, and head with easily attached ropes.

A novel capture matrix utilizing human serum albumin (HSA) and streptococcal Protein G (PG), which possesses an albumin binding domain (ABD), was used to immobilize antibodies for improved bacterial capture efficiency in immunoassays. Enzyme linked immunosorbent assays (ELISA) were used to characterize and optimize a specific protocol for the HSA-PG capture matrix; which revealed several critical factors that should be considered. The Fc binding domain, on PG, should have high affinity for the species of capture antibody used in the assay. Goat and rabbit species antibodies bound strongly to the Fc binding domain of PG. Displacement of the capture antibody, by the detector antibody should be avoided to reduce background signals. The Fc binding domain on PG should have equivalent or lower affinity for the detector antibody, when compared to the capture antibody. Goat species antibody, used as a detector antibody, did not displace the same-species capture antibody. ELISA analysis showed detection of Escherichia coli O157:H7 cells at 1.0 x 104 CFU/ml using HSA-PG and goat antibody raised against Escherichia coli O157:H7; unlabeled antibody was used for capture while HRP labeled antibody was used for detection. Studies were performed on an automated fiber optic biosensor, RAPTOR, which was used for the rapid detection of pathogens. Biosensor assays showed detection of E. coli O157:H7 at 1.0 x 10³ CFU/ml in PBS and 1.0 x 105 CFU/ml in homogenized ground beef supernatant. Capture efficiency of the HSA-PG capture matrix was studied using the biosensor and GFP-E. coli O157:H7. The amount of cells captured was less than one percent of the sample concentration. This limit of detection and capture efficiency was comparable to the streptavidin-biotin capture matrix.

Η βιοαιθανόλη παράγεται από την αλκοολική ζύμωση σακχαρούχων, αμυλούχων και κυτταρινούχων πρώτων υλών. Η χρήση της ως εναλλακτικό καύσιμο (βιοκαύσιμο), μπορεί να συμβάλει σημαντικά στην μείωση της ατμοσφαιρικής ρύπανσης. Στην βιοτεχνολογική παραγωγή αιθανόλης έχει μελετηθεί εκτεταμένα η χρήση ακινητοποιημένων κυττάρων, ως μέσο για τη αύξηση της παραγωγικότητας των ζυμώσεων. Στην παρούσα εργασία μελετήθηκε η προοπτική αξιοποίησης δύο νέων φυσικών υλικών, ως φορέων ακινητοποίησης των κυττάρων της ζύμης για την παραγωγή βιοαιθανόλης. Ο φορείς αυτοί είναι η ελίφη που αποτελεί το εσωτερικό του αποξηραμένου καρπού του φυτού Luffa cylindrica και τεμάχια του βλαστού του φυτού Arundo donax (καλάμι). Σε όλα τα πειράματα με βιοαντιδραστήρες διαλείποντος έργου, παρουσία των φορέων η παραγωγικότητα σε αιθανόλη αυξήθηκε σημαντικά και μάλιστα σε ορισμένες περιπτώσεις διπλασιάστηκε σε σχέση με τις αντίστοιχες ζυμώσεις απουσία του φορέα. Η βελτίωση είναι ιδιαίτερα σημαντική σε διαλύματα υψηλής αρχικής πυκνότητας σακχάρων (> 18 oBe), στα οποία τα κύτταρα της Saccharomyces cerevisiae, απουσία του φορέα αδυνατούν να αξιοποιήσουν το μεγαλύτερο ποσοστό των διαθέσιμων σακχάρων. Η θετική επίδραση των φορέων, παρουσίασε σταθερότητα κατά την χρήση των ίδιων φορέων σε διαδοχικές ζυμώσεις, γεγονός που αποτελεί ένα επιπλέον θετικό στοιχείο που αφορά στη διαχρονική χρήση τους. Η ακινητοποίηση των κυττάρων στους φορείς, μελετήθηκε με παρατή¬ρηση σε ηλεκτρονικό μικροσκόπιο σάρωσης (SEM). Επιβεβαιώθηκε πώς η επιφάνεια των φορέων καλύφθηκε από ένα εκτεταμένο βιολογικό υμένιο βιοκαταλύτη, το οποίο αυξάνει σημαντικά και με μικρό κόστος την παραγωγικότητα του βιοαντιδραστήρα. Συμπερασματικά, τόσο η ελίφη, όσο και το καλάμι, φαίνεται ότι συγκεντρώνουν σημαντικά πλεονεκτήματα για να αποτελέσει η χρήση τους μια ουσιαστική πρόταση για βελτίωση της τεχνολογίας παραγωγής βιοαιθανόλης. / Bioethanol is produced by the alcoholic fermentation process of sugar-containing, starchy and cellulosic raw materials. Its use as an alternative fuel (biofuel) could significantly contribute in air pollution reduction. Considerable amount of research has been carried out to develop new, highly efficient fermentation processes. One such area that has received great attention has been the use of immobilized cell systems. In the present study, we investigated the perspective to use two new natural materials as carriers for yeast cell immobilization. These materials are loofa sponge, obtained from the matured dried fruit of Luffa cylindrica and fragments of the stalk of Arundo donax (cane). All batch fermentations that carried out in the presence of carriers, had enhanced productivities (even 100% increase related to the fermentations without the carrier). This was more intense at high sugar densities (18 oBe). The positive effect of the carriers showed significant viability, when the same carriers were used in repeated batch fermentations. Cell immobilization on the carriers, was confirmed after observation in Scanning Electron Microscope. Carrier’s surface was covered by layers of biocatalyst (biofilm). Consequently, it was demonstrated that both loofa and cane, assemble enough advantages in order to constitute an effective suggestion, to improve the alcohol production technology.

Rhodococcus has diverse metabolic capabilities, such as delaying ripening of certain climacteric fruit. Nitrile hydratase (NHase), amidase, 1-aminocyclopropane-1-carboxylate deaminase (ACC deaminase), cyanidase, and β-cyanoalanine synthase-like enzyme (βCAS-like) are possibly involved in fruit ripening. The activity of these enzymes in Rhodococcus rhodochrous DAP 96253 cells were induced with selected multiple inducers (i.e. cobalt and urea). This research showed that the supplementation of selected sugars, i.e. trehalose and maltodextrin in growth media and storage buffers of R. rhodochrous DAP 96253 affected activity and stability of the enzymes mentioned above. Thermostability and osmostability of the five enzymes in whole cells (plate grown and fermented) were evaluated in this study, i.e. βCAS-like was more stable than the other four enzymes in storage conditions. Immobilized biocatalysts have practical advantages over the use of “free” whole cells. Immobilization of whole rhodococcal cells (plate grown and fermented) was employed, using techniques such as glutaraldehyde-polyethylenimine (GA-PEI) cross-linking, waxing and calcium-alginate entrapment. The GA-PEI immobilized catalysts were non-replicating and more stable in storage conditions than the catalysts produced by the other two methods. Wax or calcium-alginate immobilized catalysts (live catalysts) showed higher enzyme activity than the GA-PEI catalyst. The effects of whole and immobilized catalysts were evaluated on delayed ripening of fruit. Both free whole cells and immobilized catalysts delayed the ripening of bananas and peaches. Delayed ripening experiments showed that the catalysts were effective in direct contact and not in contact with fruit. Moreover, both free whole cells and immobilized catalysts showed antifungal activity against Aspergillus niger and Penicillium spp. Gas chromatography was performed to analyze volatile interactions between the biocatalysts and fruit. This analysis revealed that cyanide in an atmosphere with ethylene was utilized by the biocatalysts. There was also less volatile production by exposed fruit (bananas) than fruit unexposed to biocatalysts, either rhodococcal immobilized catalysts or live whole cells (plate grown and fermented).

Brachytherapy is an important method of breast cancer treatment; however, improvements in both treatment planning and delivery are needed. The procedure involves insertion of catheters in the tumor site, which, in current practice, is prone to clinically significant error. In order to improve on contemporary catheter placement accuracy, integration of pre-operative imaging, supplemented by computerized surgical planning and mathematical optimization were used to develop and test an intra-operative immobilization and catheter guidance system. A custom-template specific to each patient with optimally-placed guide-holes for catheter insertion was designed and fabricated for use on phantom studies. Template creation is based on a virtual reality reconstruction of the patient's anatomy from computed tomography imaging. The template fits on the patient's breast, immobilizing the soft tissue, to provide pre-planned catheter insertion holes for guidance to the tumor site. Agar-based phantom and target models were used for quantitative validation of the template using computed tomography imaging for template planning and validation. Planned catheter tracks were compared to post-insertion image data and distance measurements from target location were used to create an error measure. Using the latest template design spanning multiple experiments resulted in a mean of 2.6 mm, 95%, CI =3.1-2.2, which is within the clinically acceptable range of 3 mm, as validated with our clinical collaborators. Validation of the brachytherapy template on phantom tissue produced clinically acceptable results. Use of a patient-specific template for breast brachytherapy is feasible and may improve the procedure accuracy and outcome. This work has been a proof-of-concept, providing evidence to support moving forward with the next phase of patient-specific breast template trials for use in brachytherapy. / Thesis (Master, Computing) -- Queen's University, 2011-10-03 15:17:07.933

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