The purpose of this study was twofold. The first was to isolate, purify, and characterize the deoxyribonucleic acid (DNA) of Azotobacter vinelandii ATCO 12837. The second was to determine if there was irreversible binding of homologous 32P labeled DNA to recipient A. vinelandii cells.

Master of Science (Dentistry) / Dimensional stability was defined by Nicholls (1977) as “the ability (of a material) to maintain accuracy over time”, and the result of loss of accuracy, “distortion”, as “the relative movement of a single point, or group of points, away from some originally specified reference position such that permanent deformation is apparent”. Maintaining dimensional stability of dental impression materials is vital if the impression cannot be cast (in stone) soon after removal from the mouth. Dental irreversible hydrocolloid (alginate) is a major dental impression material used worldwide in many clinical procedures. However, alginate is dimensionally unstable and changes its dimensions (suffers “distortion”) after removal from the mouth. With storage times of more than ten minutes, alginate begins to distort, and after one to three hours (depending on the product and storage conditions) cannot be used for many clinical purposes, especially fixed prosthodontics such as crowns and bridges (Hampson 1955, Skinner & Hoblit 1956, Wilson & Smith 1963, Rudd et al. 1969, Miller 1975, Inohara 1977, Schoen et al. 1978, Coleman et al. 1979, Linke et al. 1985, Habu et al. 1986, Peutfeldt & Asmussen 1989, Mathilde & Peters 1992, Khan & Aziz Sahu 1995, Eriksson et al. 1998, Schleier et al. 2001, and Donovan & Chee 2004). This loss of accuracy, due to dimensional instability, manifests as a time-dependent distortion of the poured stone cast, and thus any prosthesis fabricated will not fit in the mouth. With the introduction of the more stable elastomers in the 1950s (Stackhouse 1970, Glenner 1997, Brown 2003) that could be stored for days if necessary, without loss of accuracy, the alginates fell out of favour for fixed prosthodontics. Recently, there has been a resurgence of interest in alginate for use in dental procedures where dimensional stability is critical (Peutzfeldt and Asmussen 1989, Eriksson et al. 1998). This in part is due to the favourable properties of alginate not found in the elastomers. Of greatest significance is that alginate hydrocolloid is hydrophilic, whereas elastomers are hydrophobic (Phillips & Ito 1958, Glenner 2004). Thus, alginate materials are able to reproduce wet oral areas with greater precision and to produce a superior "fit" of, say, a gold casting produced by the Lost Wax technique (Skinner and Phillips 1982). A number of reports have been published which investigate newer alginate materials that are claimed to be more dimensionally stable than older formulations. Puetzfeldt and Asmussen (1989) found that a newer alginate , if stored at 100% relative humidity, retained accuracy over 24 hours that was equivalent to that of the elastomers. More recently, the manufacturer of another alginate has claimed equivalent dimensional stability to the elastomers for up to 100 hours, and, whilst this claim has not been reported on in the literature, the present thesis will show that, under favourable conditions of storage, the material maintained clinically useful accuracy for up to 100 hours. Another approach to improving the accuracy of alginate impressions has been to combine reversible hydrocolloid with alginate (the “Bilaminar” technique). Frederick and Caputo (1997) confirmed that the new agar reversible hydrocolloids are just as accurate (at the time of removal from the mouth) as the new elastomers. Mathilde et al. (1992) and Eriksson et al. (1998) have shown that several of the “bilaminar” impression techniques for fixed prosthodontics, where alginate is used as a tray material supporting a reversible hydrocolloid (agar) wash, are as accurate and dimensionally stable as elastomers for up to three hours. However, these studies are difficult to interpret due to lack of uniformity in the testing methods, and the fact that there is no regulatory standard available to measure dimensional stability for dental alginates. The International Standard (IS) for alginate impression materials (ISO 1563:1990E) contains no specification for dimensional stability, and thus places no requirement for manufacturers to state dimensional stability properties on their labels. In contrast, ISO 4823:1992(E) specifies the IS for elastomeric dental impression materials, and it does specify a requirement for dimensional stability (less than 1.5% distortion after 24 hours). Further, the IS sets a method for determination of dimensional stability. Briefly, this method (the Optical Method) uses a travelling optical microscope to measure the accuracy of the distance between score lines on an impression of a test grid, at various time periods. The American Dental Association Specification No. 19 for dental elastomeric impression materials is identical to the IS. There is currently no specific Australian Standard (AS) for the dimensional stability of any dental impression material. Overview of Experimental Methods A. The Optical Method The aim of Part A of this investigation was to: 1. Adapt the Optical Method of the IS for elastomers to be reproducible for dental alginates. This was achieved by using a perforated test tray (to simulate clinical conditions), and measuring the grid pattern on a dental stone button after casting the test impression, rather than direct measurement of the impression, as for the IS. 2. To measure and rank the dimensional stability of a number of locally available dental alginates. Measurements of the test stone buttons proved reproducible, and the results were different for each sample, allowing them to be ranked according to dimensional stability after 50 and 100 hours of storage. The results show that the traditional optical method for measuring dimensional stability, as specified in the IS for dental elastomers, can be adapted to measure the dimensional stability of dental alginates However, the Optical Method of measuring dimensional stability of dental alginates is cumbersome and time-consuming. It was hypothesised that dimensional stability of dental alginates could be measured more conveniently by finding a thermal property that is directly proportional to dimensional stability. This method could be useful for the rapid determination of relative performance, and allow comparison with a determined benchmark. B. The Thermal Method Recently, modern methods of Thermal Analysis, Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) have been used to rapidly age various polymers, including food alginates (Chinachoti 1996), in order to measure thermal stability. This thesis shows that thermal stability is an indicator of dimensional stability. The aim of Part B of this investigation was therefore to adapt thermal analysis techniques to dental alginates, and develop a method to measure their thermal stability. These results were then compared with those for dimensional stability measured by the Optical Method to determine the relationship between thermal and dimensional stability for dental alginates. The results show that current thermal analysis methods of TGA and DSC can be adapted to measure relative dental alginate dimensional stability, and are both rapid and convenient. This study also provides evidence that commercial products differ as regards the property of dimensional stability, and can be ranked accordingly. C. Practical Application of the Methods The aim of part C of this thesis was to validate the methods (both optical and thermal) developed in this study by using them to investigate the effect of varying the water/powder ratio on the dimensional stability of dental alginates. It was shown that dimensional stability is affected by changes to the recommended water/powder ratio, that both the methods detected and measured the changes, and that the results were proportional, in that any percentage change detected by the optical method, was mirrored by the thermal method, confirming that the more convenient thermal methods can be used to measure dimensional stability.

Buprenorphine is a promising medication for the treatment of opiate abuse. The pharmacology of buprenorphine has been studied in vitro using radioligand binding and [³⁵S]GTPγS assays, and in vivo using assays of antinociception in rodents. A number of compounds with potential similar pharmacology have also been characterised. These are an iso-morphinan pyrrolidine derivative, and long-lasting 14-aminomorphinones and codeinones, in particular clocinnamox (C-CAM), a pure μ-antagonist and methoclocinnamox (MC-CAM), which has some agonist properties.

Master of Science (Dentistry) / Dimensional stability was defined by Nicholls (1977) as “the ability (of a material) to maintain accuracy over time”, and the result of loss of accuracy, “distortion”, as “the relative movement of a single point, or group of points, away from some originally specified reference position such that permanent deformation is apparent”. Maintaining dimensional stability of dental impression materials is vital if the impression cannot be cast (in stone) soon after removal from the mouth. Dental irreversible hydrocolloid (alginate) is a major dental impression material used worldwide in many clinical procedures. However, alginate is dimensionally unstable and changes its dimensions (suffers “distortion”) after removal from the mouth. With storage times of more than ten minutes, alginate begins to distort, and after one to three hours (depending on the product and storage conditions) cannot be used for many clinical purposes, especially fixed prosthodontics such as crowns and bridges (Hampson 1955, Skinner & Hoblit 1956, Wilson & Smith 1963, Rudd et al. 1969, Miller 1975, Inohara 1977, Schoen et al. 1978, Coleman et al. 1979, Linke et al. 1985, Habu et al. 1986, Peutfeldt & Asmussen 1989, Mathilde & Peters 1992, Khan & Aziz Sahu 1995, Eriksson et al. 1998, Schleier et al. 2001, and Donovan & Chee 2004). This loss of accuracy, due to dimensional instability, manifests as a time-dependent distortion of the poured stone cast, and thus any prosthesis fabricated will not fit in the mouth. With the introduction of the more stable elastomers in the 1950s (Stackhouse 1970, Glenner 1997, Brown 2003) that could be stored for days if necessary, without loss of accuracy, the alginates fell out of favour for fixed prosthodontics. Recently, there has been a resurgence of interest in alginate for use in dental procedures where dimensional stability is critical (Peutzfeldt and Asmussen 1989, Eriksson et al. 1998). This in part is due to the favourable properties of alginate not found in the elastomers. Of greatest significance is that alginate hydrocolloid is hydrophilic, whereas elastomers are hydrophobic (Phillips & Ito 1958, Glenner 2004). Thus, alginate materials are able to reproduce wet oral areas with greater precision and to produce a superior "fit" of, say, a gold casting produced by the Lost Wax technique (Skinner and Phillips 1982). A number of reports have been published which investigate newer alginate materials that are claimed to be more dimensionally stable than older formulations. Puetzfeldt and Asmussen (1989) found that a newer alginate , if stored at 100% relative humidity, retained accuracy over 24 hours that was equivalent to that of the elastomers. More recently, the manufacturer of another alginate has claimed equivalent dimensional stability to the elastomers for up to 100 hours, and, whilst this claim has not been reported on in the literature, the present thesis will show that, under favourable conditions of storage, the material maintained clinically useful accuracy for up to 100 hours. Another approach to improving the accuracy of alginate impressions has been to combine reversible hydrocolloid with alginate (the “Bilaminar” technique). Frederick and Caputo (1997) confirmed that the new agar reversible hydrocolloids are just as accurate (at the time of removal from the mouth) as the new elastomers. Mathilde et al. (1992) and Eriksson et al. (1998) have shown that several of the “bilaminar” impression techniques for fixed prosthodontics, where alginate is used as a tray material supporting a reversible hydrocolloid (agar) wash, are as accurate and dimensionally stable as elastomers for up to three hours. However, these studies are difficult to interpret due to lack of uniformity in the testing methods, and the fact that there is no regulatory standard available to measure dimensional stability for dental alginates. The International Standard (IS) for alginate impression materials (ISO 1563:1990E) contains no specification for dimensional stability, and thus places no requirement for manufacturers to state dimensional stability properties on their labels. In contrast, ISO 4823:1992(E) specifies the IS for elastomeric dental impression materials, and it does specify a requirement for dimensional stability (less than 1.5% distortion after 24 hours). Further, the IS sets a method for determination of dimensional stability. Briefly, this method (the Optical Method) uses a travelling optical microscope to measure the accuracy of the distance between score lines on an impression of a test grid, at various time periods. The American Dental Association Specification No. 19 for dental elastomeric impression materials is identical to the IS. There is currently no specific Australian Standard (AS) for the dimensional stability of any dental impression material. Overview of Experimental Methods A. The Optical Method The aim of Part A of this investigation was to: 1. Adapt the Optical Method of the IS for elastomers to be reproducible for dental alginates. This was achieved by using a perforated test tray (to simulate clinical conditions), and measuring the grid pattern on a dental stone button after casting the test impression, rather than direct measurement of the impression, as for the IS. 2. To measure and rank the dimensional stability of a number of locally available dental alginates. Measurements of the test stone buttons proved reproducible, and the results were different for each sample, allowing them to be ranked according to dimensional stability after 50 and 100 hours of storage. The results show that the traditional optical method for measuring dimensional stability, as specified in the IS for dental elastomers, can be adapted to measure the dimensional stability of dental alginates However, the Optical Method of measuring dimensional stability of dental alginates is cumbersome and time-consuming. It was hypothesised that dimensional stability of dental alginates could be measured more conveniently by finding a thermal property that is directly proportional to dimensional stability. This method could be useful for the rapid determination of relative performance, and allow comparison with a determined benchmark. B. The Thermal Method Recently, modern methods of Thermal Analysis, Thermal Gravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) have been used to rapidly age various polymers, including food alginates (Chinachoti 1996), in order to measure thermal stability. This thesis shows that thermal stability is an indicator of dimensional stability. The aim of Part B of this investigation was therefore to adapt thermal analysis techniques to dental alginates, and develop a method to measure their thermal stability. These results were then compared with those for dimensional stability measured by the Optical Method to determine the relationship between thermal and dimensional stability for dental alginates. The results show that current thermal analysis methods of TGA and DSC can be adapted to measure relative dental alginate dimensional stability, and are both rapid and convenient. This study also provides evidence that commercial products differ as regards the property of dimensional stability, and can be ranked accordingly. C. Practical Application of the Methods The aim of part C of this thesis was to validate the methods (both optical and thermal) developed in this study by using them to investigate the effect of varying the water/powder ratio on the dimensional stability of dental alginates. It was shown that dimensional stability is affected by changes to the recommended water/powder ratio, that both the methods detected and measured the changes, and that the results were proportional, in that any percentage change detected by the optical method, was mirrored by the thermal method, confirming that the more convenient thermal methods can be used to measure dimensional stability.

Pancreatic and prostate cancer are both prevalent cancers in the United States with pancreatic being one of the most aggressive of all cancers and prostate cancer being one of the most common, ranking as the number one cancer in men. Treatment of both cancers can be quite challenging as the anatomy of the pancreas and prostate, as well as the development and diagnosis of the disease can greatly limit treatment options. Therefore, it is necessary to develop new cancer treatments to help manage and prevent these cancers. Irreversible electroporation is a new non-thermal focal ablation therapy utilizing short, pulsed electric fields to damage cell membranes leading to cell death. The therapy is minimally invasive, involving the insertion of needle electrodes into the region of interest and lasts less than two minutes. Heat sink effects that thermal therapies experience near large blood vessels do not affect irreversible electroporation. This allows the treatment to be used on tumors near vasculature as well as critical structures without harming these vital regions. While irreversible electroporation is a promising new cancer therapy, further developments are necessary to improve treatment planning models. This work aims to further understand the electric field thresholds necessary to kill different types of cancer cells with a focus on pancreatic and prostate cancer. The work is done using an in vitro tumor (hydrogel) model as this model is better than traditional cell suspension studies, with added benefits over the immediate use of tissue and animal models. / Master of Science

Irreversible electroporation (IRE) is a focal ablation technique that has been shown in recent clinical trials to be effective in treating pancreatic cancer. The technique uses short, high voltage pulses to induce nanoscale pores in the target cell membranes, leading to cell death. Due to its non-thermal mechanism, IRE is particularly well suited for treating a tumor that is unresectable due to its close location to crucial structures such as blood vessels and nerves. Predicting the region of treatment is critical for optimal treatment of the tumor. The only predictive tools clinicians currently rely on for IRE treatment planning are computer tomography (CT), ultrasound (US) imaging, and real-time resistance measurement is used to monitor treatment progress. However, there is currently no method to plan optimal pulse parameters such as voltage, pulse duration, pulse number, and electrode spacing prior to treatment. Computational treatment planning models aim to perform this prediction in 3D, however, the electric field region relies on the electrical response of human tissue during IRE. This work quantifies this response for the first time and implements human tissue properties in a patient-specific, 3D treatment planning model. / Master of Science / Pancreatic cancer results in 40,000 deaths every year in the U.S, making it one of the most challenging diseases to treat. The current treatments for this disease fall short and have failed to significantly extend patient life expectancy. A technique called irreversible electroporation (IRE) has been shown in recent clinical trials to be effective in treating pancreatic cancer. IRE excels at treating tumors that are located near important blood vessels, nerves, and other important structures. However, clinicians do not have a way to visualize the region of treatment before surgery. In the research setting, 3D computational models aim to predict this area, but so far these models have been based on animal tissue, often of the incorrect organ type. This work applies IRE to human tissue samples, quantifies its electrical behavior, and implements that information in a personalized, predictive 3D model.

Osteosarcoma (OS) is the most common primary bone tumor with an annual incidence rate of 3-4 individuals per million particularly affecting children and young adults. The 5-year survival rate stands at 60-80% with the current standard of care for human OS patients who do not have metastatic disease at presentation, but this drops to 20% for patients with metastatic disease which frequently occurs in the lungs. OS is much more common in canines, with metastasis being the major contributor to mortality, the same as in humans. Metastatic OS warrants novel treatment strategies to improve prognosis and survival. High-frequency irreversible electroporation (H-FIRE) is a promising, non-thermal, minimally invasive technique that induces cell death by applying pulsed electric fields in targeted regions, potentially triggering an anti-tumor immune response that could also target and prevent metastases. Such a dual functionality of H-FIRE is uniquely suited to treat pulmonary metastatic OS. The goal of this thesis was to study the ablative and immunomodulatory effects of H-FIRE on OS in-vitro with the overall hypothesis that H-FIRE completely ablates OS cells, induces the release of damage-associated molecular patterns (DAMPs), and promotes pro-inflammatory immune activating signatures in macrophages and T cells. Using an in-vitro model, my master's thesis focused on 1) Determining the electric field strength that completely ablates OS cells 2) Evaluating the immunomodulatory effects of H-FIRE by co-culturing H-FIRE treated OS cells with macrophages and T cells separately. Our study has utilized murine, canine, and human OS and immune cells, thus demonstrating a unique cross-species approach, 3) Evaluating DAMPs (ATP, calreticulin, and HMGB1) post-H-FIRE ablation of human OS cells. Overall, our study showed that H-FIRE successfully ablated OS cells in-vitro, induced the release of DAMPs from treated cells, and promoted activation signatures in immune cells. This thesis provides foundational data for future investigations developing H-FIRE as an immunomodulatory strategy for treating metastatic OS. / Master of Science / Osteosarcoma (OS) is the most common primary bone tumor that majorly affects young adults and children with an incidence rate of 3-4 individuals per million per year. When metastasis occurs (i.e. OS spreads from its site of origin to other organs in the body), most frequently to the lungs, patients experience poor chances of recovery and survival. Currently, the treatment protocol followed for patients with metastatic OS largely includes complete surgical removal and chemotherapy both of which can be very grueling for patients. No significant improvement in the overall 5-year survival rate with current mainstay treatment has led to the urgent need of novel treatment modalities for treating patients with pulmonary metastatic OS. High-Frequency Irreversible Electroporation (H-FIRE) is a novel non-thermal tumor ablation strategy that utilizes electrical pulses to create pores on the cell membrane, thus leading to irreversible damage and cell death. These dying tumor cells release certain molecules and proteins that send danger signals to activate the body's own immune system against the tumor. H-FIRE with its dual function of destroying the targeted tumor region via electroporation and distant metastases via activating immune system is uniquely suited to treat pulmonary metastatic OS. This thesis is the first to investigate H-FIRE ablation and immunomodulation for OS. We hypothesized that H-FIRE can completely destructs OS cells, promotes the release of danger signals, and causes immune activation. Using an in-vitro model, this thesis focused on 1) Determining the electric field strength needed for complete OS cell destruction by H-FIRE 2) Evaluating the immune activation potential of H-FIRE by exposing these H-FIRE treated cells to immune cells like macrophages and T cells separately. We utilized human, mouse, and dog-derived OS cells to increase the biological and clinical relevance of our study. 3) Evaluating certain proteins that act as danger signals post-H-FIRE treatment of human OS cells. Overall, our results indicated that H-FIRE can successfully destruct OS cells in-vitro, promotes the release of danger signals, and induces immune activation. This thesis contributes to providing crucial preliminary data in the development of H-FIRE as a novel ablation and immunomodulation treatment strategy for pulmonary metastatic OS.

Breast cancer (BC) is the second most common cause of cancer-related deaths for women in the United States, estimated to affect 1 in 8 women. Difficulties arise in BC treatment due to the hormone sensitivity and heterogeneity of the malignancies, and the poor prognosis after metastases. Due to the immense physical and psychological effects of conventional surgical methods, minimally invasive, non-thermal, focal electroporation-based ablation therapies are being investigated for the treatment of BC. Irreversible Electroporation (IRE) delivers a series of long, monopolar electrical pulses via electrodes inserted directly into the targeted tissue which disrupt cellular membranes by creating nano-scale pores, killing the cells via loss of homeostasis while promoting an immune response. However, IRE requires cardiac synchronization and a full-body paralytic to mitigate unwanted muscle contractions, which motivated the creation of second generation High-Frequency IRE or H-FIRE. H-FIRE delivers short, bipolar pulses to destroy cancer cells without muscle contractions and nerve excitation, and allows for more tunable treatment parameters. Throughout my thesis, I discuss investigations of H-FIRE for the treatment of triple-negative and hormone-sensitive BC cell lines and compare efficacy to IRE outcomes. To further establish the translation and understanding of H-FIRE for BC applications, my master's thesis focuses on: (1) determining the lethal electric field threshold of both cell lines in a 3D hydrogel matrix after H-FIRE and IRE; and (2) employ those values in a single bipolar probe numerical model to simulate in vivo treatments. The culmination of this thesis advances the use of H-FIRE in breast tissues, as well as demonstrates how in vitro data can be used to develop clinically relevant numerical models to better predict in vivo treatment outcome. / Master of Science / Breast cancer (BC) is one of the most deadly forms of cancer for women in the United States, affecting every 1 in 8 women. Difficulties arising in the treatment of BC include the hormone sensitivity of malignancies, metastatic tendencies, and the diversity of the tissue that characterizes the breast. Surgical options like mastectomy or lumpectomy are most often used when treating BC; however, these are incredibly taxing on the patient. This reason has sparked investigations of focused ablation modalities for the treatment of BC, specifically non-thermal mechanisms like electroporation-based therapies. Electroporation explains the phenomenon that cells subjected to a high enough electric field will result in increased membrane permeability, allowing for the entrance of therapeutic agents in reversible mechanisms, or cell death beyond an irreversible point. Irreversible Electroporation (IRE) has shown success for the treatment of prostate, liver, kidney, and pancreas. However, due to some drawbacks, second generation High-Frequency IRE (H-FIRE) is increasingly being investigated for certain cancer types and is the main focus of this thesis project. Within this thesis, I discuss investigations of H-FIRE with applications to treat malignant breast cell lines. Specifically, my thesis focuses on: (1) determining the point at which cancer cells damage is irreversible; and (2) incorporate those values into a numerical model used to simulate electroporation treatment if a tumor were embedded in a layer of fatty connective breast tissue. The culmination of this thesis enhances our understanding of H-FIRE in the breast, with the hopes of future transition of application into animal studies and ultimately the clinic.

Typescript (photocopy). / Digitized by Kansas Correctional Industries

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.