<p> Temperature-sensitive mutants of the bacteriophage PBS 2 were isolated from lysates treated with various mutagens. Complementation tests assigned the mutants to 10 cistrons. The mutants were mapped by two factor crosses and formed a linear map approximately 50 recombination percent in length. </p> <p> A method for phage transformation was developed. By the use of wild type DNA fragments fractionated according to their guanine + cytosine content in HgCs₂SO₄ gradients, it was possible to determine the base composition of certain regions of the chromosomes. </p> / Thesis / Doctor of Philosophy (PhD)

<p> Temperature sensitive microgel latexes of poly (N-isopropylacrylamide) cross-linked with N-N'methylene bisacrylamide (BA) were prepared and characterized by Dynamic Light Scattering, Titration and Electrophoresis. The study of gels, including temperature sensitive gels, is limited by the large size of traditional bulk gels which are slow to respond to changes and are difficult to measure. An alternative system, which may be easier to study, is a microgel latex which would constitute small particles of gel which would respond quickly to changes in their environment and could also be measured using colloidal measuring techniques like dynamic light scattering and particle electrophoresis.</p> <p> Monodisperse and stable microgel latex particles were prepared by reacting Nisopropylacrylamide (NIPAM) monomer with a cross-linking agent BA in water at 70°C with a surfactant (sodium dodecylsulfate) present. Latexes prepared without surfactant were polydisperse.</p> <p> Characterization of the poly (NIPAM) particles by dynamic light scattering at several different temperatures showed that the particles go through a transition from a water swollen gel at low temperature to a shrunken gel with a low water content at high temperature. The transition occurs about 32°C. The degree of swelling of the poly (NIPAM) particles can be expressed by the Flory-Huggins Interaction parameter c and is dependent upon the level of cross-linking agent included.</p> <p> Titration and electrophoresis results indicate that the particles contain about 0.39 Coulombs per gram of polymer of carboxylic and sulfuric charged end groups which are distributed throughout the particle.</p> / Thesis / Master of Engineering (MEngr)

<p>Mild hyperthermia (39°C-43°C) has numerous therapeutic benefits as an adjuvant therapy in the treatment of a variety of tumor types. Hyperthermia increases tumor blood flow and vascular permeability, promoting drug delivery and tumor oxygenation. Hyperthermia enhances the uptake and efficacy of numerous chemotherapeutic agents, including cisplatin, resulting in increased cytotoxicity. In addition to these biological responses, hyperthermia can be used as a drug-release trigger for temperature-sensitive nanoparticles, resulting in an improved and more targeted drug delivery system. Cisplatin was chosen because 1) it shows broad spectrum activity against a wide range of heatable cancers (i.e., those in sites such as the pancreas, colon and rectum, cervix and bladder, and 2) the same hyperthermic temperatures that enable temperature-sensitive lipsome-drug release also enhance cisplatin-induced cytotoxicity.</p><p>The role of hyperthermia in enhancing cisplatin delivery and cytotoxicity was investigated at both the cellular and tissue levels. While hyperthermia treatment is applicable to a variety of tumor types, the focus of this work was on bladder cancer. The synergistic effects of hyperthermia and cisplatin were investigated, along with the role of copper transport protein 1 (Ctr1) in this process. In addition, cisplatin was encapsulated within temperature-sensitive liposomes, which were used in combination with hyperthermia for targeted drug delivery. These studies demonstrated that the combination of cisplatin and hyperthermia improved drug delivery, and potentially anti-tumor efficacy, and that targeted delivery was enhanced through incorporation of temperature-sensitive liposomes. As many current methods for administering bladder hyperthermia have drawbacks, such as invasiveness and regional heating, the final aim of this study was to develop and test a less-invasive and more focused preclinical bladder heating device in a rat model. </p><p>Hyperthermia sensitizes cells to the cytotoxic effects of the commonly used chemotherapeutic agent cisplatin by increasing drug accumulation and subsequent platinum-DNA adduct formation. However, the molecular mechanisms underlying this enhancement remain unclear. Understanding the fundamental mechanisms involved in the synergistic interaction is necessary to increase the therapeutic benefits of this combination in the clinic. The synergism between the anti-cancer benefits of cisplatin and the drug delivery benefits of hyperthermia may offer a novel and more effective treatment for many cancer patients. We hypothesized that hyperthermia increases cisplatin accumulation and efficacy in part by modulating the function of Ctr1, a major regulator of cellular cisplatin uptake. To test this hypothesis, we examined the significance of Ctr1 during combined hyperthermia and cisplatin therapies and assessed the importance of cisplatin- and hyperthermia-induced Ctr1 multimerization in enhancing cisplatin cytotoxicity. We observed increased Ctr1 multimerization following hyperthermia treatment (41°C) in vitro, compared to normothermic controls (37°C), suggesting that this may be a mechanism for increased cisplatin uptake in heat-treated cells. The impact of increased Ctr1 multimerization was evaluated by measuring platinum accumulation in wild-type (WT) and Ctr1-/- cells. WT cells contained greater levels of platinum compared to Ctr1-/- cells. A further increase in platinum was observed following hyperthermia treatment, but only in the WT cells. Hyperthermia enhanced cisplatin-mediated cytotoxicity in WT cells with a dose-modifying factor (DMF) of 1.8 compared to 1.4 in Ctr1-/- cells. Our data suggest that heat increases Ctr1 activity by increasing multimerization, resulting in enhanced drug accumulation. Although we recognize that the effect of heat on cells is multi-factorial, our results support the hypothesis that Ctr1 is, in part, involved in the synergistic interaction observed with cisplatin and hyperthermia treatment. </p><p>In addition to assessing cisplatin delivery at the cellular level, we evaluated cisplatin delivery at the tissue level, using novel cisplatin-loaded temperature-sensitive liposomes. We hypothesized that delivering cisplatin encapsulated in liposomes under hyperthermic conditions would improve the pharmacokinetic profiles of cisplatin, increase drug delivery to the tumor, decrease normal tissue toxicity, and enhance the anti-tumor activity of cisplatin. We successfully prepared temperature-sensitive liposomes loaded with cisplatin and demonstrated that heat (42°C) sensitizes cisplatin-resistant cells to the cytotoxic effects of cisplatin in vitro. </p><p>Decreased toxicity was observed in animals treated with the cisplatin liposome (± heat) compared to the free drug treatments. A pharmacokinetic study of cisplatin-loaded temperature-sensitive liposomes and free drug was performed in tumor-bearing mice under normothermic and hyperthermic conditions. Cisplatin half-life in plasma was increased following liposome treatment compared to free cisplatin, and cisplatin delivery to the tumors was greatest in mice that received liposomal cisplatin under hyperthermia. These initial in vivo data demonstrate the potential effectiveness of this cisplatin-loaded liposome formulation in the treatment of certain types of cancer. To assess the anti-cancer efficacy of the liposome treatment, a tumor growth delay study was conducted and demonstrated equivalent efficacy for the cisplatin-loaded temperature-sensitive liposome compared to free drug. </p><p>In addition to the liposome work, we developed and evaluated a novel heating device for the bladder. Despite the evidence that hyperthermia is an effective adjuvant treatment strategy, current clinical heating devices are inadequate, warranting the development of a new and improved system. We induced hyperthermia using ferromagnetic nanoparticles and an alternating magnetic field device developed by Actium Biosystems. Initial preclinical studies in a rat model demonstrated preferential bladder heating. However, our preliminary studies show severe toxicity with the direct instillation of the nanoparticles in the bladder, and further studies are needed to potentially modify the nanoparticle coating, the catheterization procedure, as well as to develop a different animal model.</p> / Dissertation

F. tularensis is a Gram-negative bacterial pathogen and it is the causative agent of tularemia. It has the ability to replicate to high numbers within a variety of host cells, including macrophages. Little is known of its virulence mechanisms; however, all species of Francisella contain a cluster of virulence genes known as the Francisella Pathogenicity Island (FPI), which is thought to encode a type 6 secretion system. While 14 of the 18 FPI genes encode products required for intracellular growth in macrophages, the functions of most of these proteins remain to be determined. Therefore, further work is required to understand the role played by the FPI in Francisella pathogenesis. In this thesis, the localization of the core FPI proteins IglA, IglB, IglC and IglD, was examined in order to further elucidate of the structure and activities of the FPI-encoded secretion system. Deletion mutagenesis of pdpA was performed to determine how host intracellular signalling might be affected by secretion of the putative FPI effector protein PdpA. In addition, variations in virulence between different biotypes of Francisella were investigated with respect to the role played by the FPI protein PdpD. Considering the highly infectious nature of Francisella and the absence of a quality vaccine, it is clear that this organism represents an excellent model for proof of principle investigations focussing on new vaccine technologies for intracellular pathogens. The second half of this thesis describes the construction and characterization of live attenuated temperature-sensitive vaccines. These vaccines were created in the intracellular pathogen F. novicida through allelic replacement of essential genes with naturally-occurring, cold-adapted, thermolabile homologues isolated from Arctic bacteria. Thus, the objectives of this work were twofold: to provide further characterization of the structural components and effector proteins associated with the FPI-encoded secretion system, and to develop a new and effective vaccine technology for use against intracellular bacteria. / Graduate

Temperature-sensitivity (TS) is often used as a way to attenuate microorganisms to convert them into live vaccines. Studies indicate that live vaccines are often necessary for the complete clearance of certain pathogenic organisms. In this work we explore the use of TS genetic circuits that express lethal genes for their potential utility as a widely applicable approach to TS attenuation. Here, we use restriction endonucleases as the lethal gene products. We tested different combinations of TS repressors and cognate promoters controlling the expression of genes encoding restriction endonucleases inserted at four different non-essential sites in the Escherichia coli chromosome. We found that the presence of the restriction endonuclease genes did not affect the viability of the host strains at the permissive temperature, but that expression of the genes at elevated temperatures killed the strains to varying extents. The location of the genetic circuit cassette in the chromosome was critical, and insertion at the ycgH site led to minimal cell death. Induction of the TS circuit in a growing culture led to a pre-mature leveling off of the optical density, and a shift in the number of cells that could exclude a dye that indicated cell viability. Incubation of cells initially grown at low temperature and then suspended in phosphate buffered saline at high temperature, led to about 100-fold loss of cell viability per day compared to minimal loss of viability for the parental strain. The Dual strain containing two different genetic circuits was found to have reduced escape frequency compared to single circuit strains. However, strains carrying either one or two TS lethal circuits could generate mutants that survived high temperature. These mutants included start codon deletions as well as upstream deletions of the TetRD1 encoding gene as well as complete deletions of the lethal gene circuits. / Graduate

A segment of transpiring wall is installed near a row of unshaped film holes. The effects on the aerodynamic performance and cooling downstream of the row of cylindrical holes in the presence of transpiration is studied numerically. The changes in behavior of the film due to relative positioning of the injection sources and blowing ratios are predicted to understand the sensitivity of cooling and aerodynamic losses on the relative positioning of the two sources and each blowing ratio. The results indicate that a coupling of the two sources allows a more efficient use of coolant by generating a more uniform initial film resulting in improved component durability through reduction of hot- streaks. With careful optimization the discrete holes can be placed farther apart laterally operating at a lower blowing ratio with a transpiration segment making the large deficits in cooling effectiveness mid-pitch less severe, overall minimizing coolant usage. Addition of transpiration increases the aerodynamic losses associated with injection. This effect can be arguably small compared to corresponding thermal benefits seen by coupling the two. Comparisons of linear superposition predictions of the two independent sources with the corresponding coupled scenario indicate the two films positively influence one another and outperform predictions. The interaction between the two films is dependent upon the relative placement of the transpiration; all relative placements have an overall beneficial effect on the cooling seen by the protected wall. An increase in area-averaged film cooling effectiveness of 300% is seen along with only a 50% increase in loss coefficient by injecting an additional 10% coolant. In this study the downstream placement of transpiration is found to perform best of the three geometries tested while considering cooling, aerodynamic losses, local uniformity and manufacturing feasibility. With further study and optimization this technique can potentially provide more effective thermal protection at a lower cost of aerodynamic losses and spent coolant. A method of measuring the local temperature of a porous wall is also discussed. Measurements are taken with temperature sensitive paint applied in thin coats to the wall. This technique was validated on a 40PPI, 7% relative density aluminum porous coupon. Measurements of discharge coefficients as well as downstream effectiveness data are included to verify the flow through the porous wall was unaltered by applying the paint. A maximum deviation in film-cooling effectiveness of 9% between the two cases with the majority of data falling within 4% was found, very similar to the experimental uncertainty of the rig. This excellent agreement between the repeated tests showed that by applying thermal paint to a wall of such porosity does not significantly affect the flow exiting the wall and hence the measurement technique can readily be applied to transpiration cooling studies at this scale. Methods of filtering the temperature sensitive paint on the porous wall are presented.

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Mycobacterium tuberculosis remains one of the most common worldwide causes of illness and death due to an infectious disease. The emergence of multiple and extreme-drug resistant strains has increased the need to find an effective vaccine for tuberculosis. The goal of our research group is to engineer a temperature-sensitive (TS) M. tuberculosis strain that can be used as a tool in vaccine development. One approach to create TS M. tuberculosis involves the integration of the essential gene ligA encoding a TS NAD+ dependent DNA ligase, which was taken from the psychrophilic organism Pseudoalteromonas haloplanktis. The integration and functioning of ligA was demonstrated in the fast-growing organism Mycobacterium smegmatis. This strain had a TS phenotype with growth limited to below 37°C. The strain was found to have a stable TS phenotype and did not mutate to a temperature-resistant form at a detectable level. Following experiments with the fast growing M. smegmatis, the integration of the ligA gene was attempted in slow-growing M. tuberculosis. Merodiploids of M. tuberculosis containing both the psychrophilic and the WT ligA gene in its chromosome were obtained. The second approach used for the development of TS M. tuberculosis was the directed evolution of native M. tuberculosis essential genes. An advantage of this approach is that the gene encoding the essential protein will resemble the native M. tuberculosis gene and thus will closely match the native transcriptional and translational rates. A system to screen and select for TS essential genes engineered by directed evolution was designed, where the essential gene on the chromosome of E. coli was knocked out and this gene was supplied on a conditionally replicating plasmid. As a first step in developing this directed evolution approach, a family of conditionally replicating plasmids were created and tested in an essential gene knock-out strain of E. coli. / Graduate

Limb haemodynamic responses during heat-stress and the importance of local vs. central temperature-sensitive mechanisms towards their regulation remain poorly understood, both at a whole-limb level and within individual tissues (i.e. skeletal muscle and skin). The aims of this thesis were to 1) investigate the haemodynamic responses at rest to direct thermal challenges both at a local level and during progressive elevations in systemic heat stress, 2) to ascertain the contribution of local vs. systemic mechanisms towards this regulation, and 3) to investigate the same responses during single-legged small-muscle mass exercise to near maximal levels. Results from Chapters 4 and 5 characterised the haemodynamic responses during isolated cooling and heating of the arm and leg, and provided evidence of alterations in both skin and skeletal muscle blood flow controlled solely through local temperature-sensitive mechanisms. While local cooling led to modest decreases in limb blood flow due to decreases in mean blood velocity alone, increases during heating occurred as a result of an increased antegrade flow, a diminished retrograde flow, and a reduction in the potentially pro-atherogenic oscillatory shear index. In Chapter 6, whole-body heating with isolated single leg cooling displayed the continued control of limb blood flow via local thermosensitive mechanisms alone, as cooled leg blood flow remained unchanged despite significant elevations in core temperature, cardiac output, and opposing heated leg blood flow. Furthermore, elevations in heated leg V̇O2 suggested a possible metabolic contribution to the observed skeletal muscle hyperaemic response. During incremental single-legged knee-extensor exercise to near maximal levels, blood flow was determined by a combination of metabolic workload and local tissue temperatures, regardless of whether systemic heat stress was present. Chapter 7 revealed that whilst skin and muscle blood flow in the leg continued to increase in line with local temperatures to levels of severe heat stress, rapid cooling of the leg when hyperthermic resulted in a similar reverse response in muscle tissues only, as skin blood flow remained elevated despite the abolition of high skin and subcutaneous temperatures. In addition, evidence was provided that moderate levels of whole-body heat stress provided little additional benefit to anti-atherogenic shear profiles than that experienced during isolated limb heating alone. Taken together, these findings suggest that local thermosensitive mechanisms dominate limb blood flow control during direct rapid heating in humans both at rest and during small muscle mass exercise, but that underlying central mechanisms may act to maintain flow when local temperatures are reduced in the face of high core temperatures.