Cancer is one of the challenges for today's medicine and therefore a great deal of effort is being put into improving known methods of treatment and developing new ones. A new method that has been proposed is magnetic hyperthermia where magnetic nanoparticles linked to the tumor dissipate heat when subjected to an alternating magnetic field and will thus increase the temperature of the tumor. This method makes the tumor more susceptible to radiation therapy and chemotherapy, or can be used to elevate the temperature of the tumor cells to cause cell death. The particles proposed for this are single core and often have a size in the range of 10 nm to 50 nm. To achieve an effective treatment the particles should have a narrow size distribution and the proper size. In this work, a theoretical model for predicting the heating power generated by magnetic nanoparticles was evaluated. The model was compared with experimental results for magnetite particles of size 15 nm to 35 nm dissolved in water. The properties of the particles were characterized, including measurements of the magnetic saturation, the effective anisotropy constant, average size and size distribution. To evaluate the results from the model the AC susceptibility and heating power were experimentally determined. The model is a two-step model. First the out-of-phase component of the AC susceptibility as a function of frequency is calculated. Then this result is used to calculate the heating power. The model gives a correct prediction of the shape of the out-of-phase component of the susceptibility but overestimates its magnitude. Using the experimentally determined out-of-phase component of the susceptibility, the model estimation of the heating power compares quite well with the measured values.

Hepatoma Tissue Culture (HTC) cells grown in the presence of 60µM arachidonic acid for 24, 36 and 48 hours became progressively more thermosensitive than control cells. However, this difference in thermal sensitivity was only detectable with the clonogenic assay and not with the colorimetric assay. Attempts were also made to manipulate cellular cholesterol levels. Firstly, some cells were incubated with phosphatidylcholine liposomes to deplete the plasma membrane of cholesterol: Secondly, another group of cells were treated with 25 hydroxycholesterol, an inhibitor of cholesterol synthesis, to lower cholesterol levels: Finally, a third group of cells were supplemented with cholesterol hemisuccinate, a hydrophilic ester of cholesterol. The first two approaches did not enhance the thermal sensitivity of HTC cells. Supplementation with cholesterol hemisuccinate, which was predicted to partition in to the plasma membrane and reduce membrane fluidity, resulted in increased thermal sensitivity of the cells. Thus, the thermal sensitivity of HTC cells could be enhanced by supplementation with either arachidonate or cholesterol hemisuccinate. A rapid plasma membrane isolation procedure was developed which generated plasma membranes in relatively high yield and purity. The plasma membrane- enriched fraction was also assayed for contaminating intracellular membranes by determining marker enzyme activities associated with these membranes. Using this method, plasma membranes were prepared from HTC cells grown m 60µM arachidonic acid for 36 hours and from cells grown in normal medium. Analysis of the plasma membrane showed that the arachidonic acid content of the phospholipid fatty acyl groups had been significantly increased in cells grown in the presence of this fatty acid. There was no change in the cholesterol/phospholipid molar ratio or cholesterol concentration relative to amount of protein in the plasma membranes from the two cell populations. The measurement of fluidity using DPH fluorescence polarisation revealed that the increase in the arachidomc acid content of the plasma membrane phospholipid acyl groups was associated with enhanced plasma membrane fluidity when compared to control plasma membranes. This increase in plasma membrane fluidity correlated with the enhanced thermal sensitivity of the cells grown in arachidonic acid-containing medium when compared to cells grown in normal medium. Furthermore, the thermal sensitivity of Na(^+), K(^+) –ATPase and alkaline phosphodiesterase I were assessed in plasma membranes derived from arachidonic acid-supplemented and control cells. The enhanced fluidity of plasma membranes derived from arachidonate-supplemented cells also correlated with increased thermosensitivity of alkaline phosphodiesterase I.

Medical hyperthermia refers to heating of tumors to temperature levels which are lethal to the cells for sufficient periods of time or rendering the cancerous cells more sensitive to ionizing radiation or chemotherapy. In order to increase the temperature in cancerous tissues, high power solid state microwave amplifiers need to be used. Recently ultra wide-band and continuous wave microwave methods have received increasing attention. Using adaptive focusing annular phase array applicators the radiation pattern can be adjusted according to tumor size and seating depth. For this purpose power amplifiers operating across 300MHz-1000MHz having a minimum output power of 150W needed to be designed. By varying the operating frequency the penetration depth can be controlled. Since currently 12 (with plans to increase the number to 18) of these amplifiers will be operating simultaneously in the designed system, the power added efficiency of the amplifier will be important both to regarding the cost of electricity and also allow for easier cooling requirements and thus a more compact system. The aim in this project is to have an efficiency of 60% across the band. In this thesis a power amplifier working in a push-pull configuration, designed using an NXP LDMOS device (BLF-647P) capable of delivering 200W RF power is demonstrated. During this thesis, different power combining topologies were studiedusing a nonlinear model that was developed in ADS using basic data provided by NXP. Using the developed model, load pull simulations have been performed and the input, two output matching networks are designed based on results from the load pull simulations. The design was manufactured and mounted on a copper base plate designed for this work which allowed efficient water cooling as well as serving as a fixture to firmly attach the matching circuits to the transistor and connectors. The measurements show that the design is capable of delivering more than 125 W from 360-940 MHz in pulsed mode operation with a mean efficiency of 50% which was measured in continuous mode. This work has demonstrated a high power wideband amplifier with high efficiency needed to drive future hyperthermia systems. The high efficiency of the amplifier allows for modern hyperthermia systems to be built in a more compact configuration with lower operating cost, which would not be possible with commercially available amplifiers.

In treating cancer, a primary consideration is the target specificity of the treatment. This is a measure of the treatment dose that the cancerous (target) tissue receives compared to the dose that healthy tissue receives. Nanoparticle (NP) based treatments offer many advantages for target specificity compared to other forms of treatment due to their ability to selectively target tumors. One benefit of using magnetic NPs is their ability to release heat, which can both sensitize tumors to other forms of treatment as well as damage the tumor. The work here aims to incorporate a broad range of relevant physics into a comprehensive model. NP aggregation is known to be a large source of uncertainty in these treatments, thus a framework has been developed that can incorporate the effects of aggregation on NP diffusion, NP heat release, temperature rise, and overall thermal damage. To quanitify thermal damage in both healthy tissue and tumor tissue, the Cumulative Equivalent Minutes at 43 textcelsius~model is used. The Pennes bioheat equation is used as the governing equation for the temperature rise and included in it is a source heating term due to the NPs. NP diffusion and aggregation are simulated via a random walk process, with a probability of aggregation determining if nearest neighbor particles aggregate at each time step. Additionally, models are developed that attempt to incorporate aggregation effects into NP heat dissipation, though each proves to only be accurate when there is little aggregation occurring. In this work, verification analyses are done for each of the above areas and, at minimum, qualitatively accurate results have been achieved. Verification results of this work show that aggregation can be neglected at concentrations on the order of $100~nM$ or less. This however only serves as a rough estimation and further work is needed to gain a better quantitative understanding of the effects of NP concentration on aggregation. Using this concentration as a limitation, results are presented for a variety of tumor sizes and concentration distributions. Because this work incorporates a variety of physics and numerical methods into a single encompassing model, depth and physical accuracy in each area (bio-heat transfer, diffusion via random walk, NP energy dissipation, and aggregation) have been somewhat limited. This does however provide a framework in which each of the above areas can be further developed and their effects examined in the overall course of treatment. / Master of Science

These studies investigated 1) mechanisms underlying the well-established reduction in orthostatic tolerance (OT) that occurs in humans during heat stress (HS) relative to normothermia (NT) with particular focus on determining factors contributing to the high degree of inter-individual variability in this phenomenon; and 2) influence of obesity on OT, and mechanisms underlying reduced OT, should it exist. In Study #1, OT was assessed during lower body negative pressure (LBNP), and quantified with a cumulative stress index (CSI). Differences in CSI (CSIdiff) between thermal conditions were used to categorize individuals most (LargeDiff) and least (SmallDiff) affected by HS (P<0.001). Cerebral perfusion [indexed as middle cerebral artery blood velocity (MCA Vm̳̳e̳a̳n̳)] was reduced during HS compared to NT (P<0.001); however, the magnitude of reduction did not differ between groups (P=0.51). In the initial stage of LBNP during HS (LBNP20), MCA Vm̳̳e̳a̳n̳ and end-tidal CO₂ (PETC̳O̳₂) were reduced, and heart rate (HR) was higher in the LargeDiff group compared to SmallDiff group (all P<0.05); yet, mean arterial pressure was similar (P=0.23) suggesting impaired mechanisms regulating MCA Vm̳̳e̳a̳n̳ may affect OT. In Study #2, mechanisms of cerebrovascular control were compared in LargeDiff and SmallDiff individuals. Although estimates of cerebral autoregulation (CA) and cerebrovascular reactivity to CO₂ were improved and reduced respectively, during HS compared to NT (all P<0.05), no relationship existed between CA or cerebral reactivity to hypocapnia and CSIdiff (all P>0.05). In Study #3, OT was lower in obese compared to non-obese individuals (P<0.01), and BMI was negatively correlated with CSI (R = -0.47; P < 0.01). HR was elevated at rest and in every level of LBNP (all P<0.05) in obese; yet, peak HR during LBNP was similar between groups (P=0.90). MCA Vm̳̳e̳a̳n̳ and cerebral vascular conductance were similar at rest and during LBNP (both P>0.05), and CA was similar between groups (P>0.05). In summary, a high HR prior to-, and a high HR and reduced MCA Vm̳̳e̳a̳n̳ at the onset of an orthostatic challenge result in reduced OT during HS in healthy individuals; however, reduced OT in obese is related to a higher %peak HR at rest. / text

Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV.

MSc., Faculty of Science, University of the Witwatersrand, 2011 / Wild animals are captured for management, health, translocation and research purposes. Capture is an unnaturally stressful event, which may result in morbidity or mortality. An attributing cause of the morbidity and mortality is capture-induced hyperthermia; the larger the magnitude and the longer the duration of this captured-induced hyperthermia, the greater the likely risk to the animal. The most common practice currently used in the field to lower body temperature is to douse hyperthermic animals with water. However, the water used is often at ambient temperatures and its efficacy is not known. We investigated whether this method and alternative methods are effective at lowering the body temperature of hyperthermic animals. To achieve these aims we implanted 19 blesbok with miniature temperature-sensitive data loggers in their abdomens and into their subcutaneous layers (at the sites of the flank, groin, lower neck and upper neck). The loggers continuously recorded core body temperatures of the blesbok throughout the study period at an interval of six minutes. We successfully retrieved complete data sets from 12 blesbok. The animals were captured on six separate occasions using a technique which elicited hyperthermia. Five animals were cooled by dousing with water of different temperatures (4°C, 17°C, 28°C) and fanning after dousing with 28°C water, in random order. Seven animals were cooled by ice packs, spraying a fine mist spray, intravenous (IV) infusion of one litre of 4°C water and 28°C water-dousing. Through the use of our continuous logging of body temperature we established the normal body temperature of the blesbok, which displayed a regular 24 hour body temperature pattern. The average daily body temperature of the blesbok was 38.8°C ± 0.4°C, with a minimum body temperature of 37.9°C ± 0.1°C and a maximum body temperature of 39.4°C ± 0.1°C. The body temperature after capture was as high as 41°C-42°C, which was significantly higher than the normal body temperature (Student’s t-test, P < 0.05). The animals were cooled once they were immobilised and the start of cooling was denoted as time zero. In the control (no active cooling) intervention the body temperature decreased to only about 40°C. Dousing animals with water, irrespective of its temperature, resulted in significant cooling (P < 0.05) of the animals, as indicated by their minimum body temperature reached, change in body temperature and rate of cooling. The water-dousing interventions decreased the body temperature to about 38°C after an hour, which was significantly lower than the control (RM-ANOVA, P < 0.05) but there was no significant difference in the minimum body temperature reached between the different water temperatures or by the addition of fanning (RM-ANOVA, P > 0.05). The water-dousing interventions cooled the animals more quickly than did the control (RM-ANOVA, P < 0.05), and the coldest water (4°) cooled the animals quicker than did the 28°C water-dousing (RM-ANOVA, P < 0.05). The core body temperature minus the subcutaneous temperature was calculated, and revealed a peak difference of about 3.5°C after the 4°C water-dousing. Ice-packs also resulted in significant cooling (P < 0.05) of the animals, as depicted by their minimum body temperature reached, change in body temperature and rate of cooling. The ice-packs lowered the body temperature to a minimum of about 38°C, which was significantly lower than the control (RM-ANOVA, P < 0.05). The ice-packs also cooled the animals significantly faster than did the control, intravenous infusion and mist spray (RM-ANOVA, P < 0.05) but cooled as quickly as the 28°C water-dousing (RM-ANOVA, P > 0.05). The core body temperature minus the subcutaneous temperature for the ice-packs peaked at a difference of about 3°C. The IV infusion and mist spray were ineffective cooling methods and did not significantly (P > 0.05) alter the minimum body temperature or rate of cooling. Even though the IV infusion caused a significant reduction in body temperature by 1°C, the cooling effect from the IV infusion was short-lived because the minimum body temperature reached after the intravenous infusion and mist spray was ultimately similar to the body temperature seen in animals receiving the control (RM-ANOVA P > 0.05). Also, the intravenous infusion and mist-spray cooled as slowly as did the control (RM-ANOVA P > 0.05). Therefore, water-dousing in this study was the most effective and practical method to cool hyperthermic blesbok. Although all the water temperatures (4°C, 17°C and 28°C) that we tested were effective, the coldest water (4°C) cooled the animals quickest. The addition of fanning to the 28°C water-dousing did not increase cooling. Ice-packs were also effective but may be not as easy to use as the water-dousing method as ice-packs are large and need to be kept frozen, and therefore are cumbersome for use in the field.

The main goal of this project was the development of carbon nanotubes as a candidate for dual-functioning magnetic hyperthermia structure and magnetic resonance imaging contrast agent. This was achieved by filling carbon nanotubes with continuous ferromagnetic α-Fe nanowires and surface functionalized with paramagnetic Gd(III). Also, length control of both nanotube and nanowire was investigated. Firstly, a low vapour flow-rate and constant evaporation temperature chemical vapour deposition method based on the thermal decomposition of ferrocene was employed which achieved continuous α-Fe nanowires on the same scale as the nanotube for lengths >10 m without the necessity of post-synthesis heat-treatment or introduction of other precursor elements. The low vapour flow-rate regime has the advantage of sustaining the intrinsic temperature gradient at the tip of the forming structure which drives the vapour feedstock to the growth front to guarantee continuous nanowire formation. For initially mixed-phase nanowires of length less than 10 μm, the continuous α-Fe nanowires were achieved by postsynthesis heat treatment. Secondly, a simple wet chemical method involving only sonication in aqueous GdCl3 solution was used for surface functionalization of iron-filled multiwalled carbon nanotubes with gadolinium. Functional groups on the sidewalls produced by the sonication provide active nucleation sites for the loading of Gd3+ ions. Characterization by electron paramagnetic resonance, electron energy loss spectroscopy, and high-resolution transmission electron microscopy confirmed the presence of Gd3+ ions on the sidewall surface. The ferromagnetic properties of the encapsulated iron nanowire maintained after surface functionalization. At room temperature a saturation magnetization of 40 emu/g and a coercivity of 600 Oe were observed. Heating functionality in an alternating applied magnetic field was quantified through the measurement of specific absorption rate: 50 W/gFe and the intrinsic loss power: 1.12 nHm²kg⁻¹ at magnetic field strength 8 kA/m and frequency of 696 kHz. These structures exhibited an extremely high relaxivity r₁ ~ 200 mM⁻¹ s⁻¹ at high magnetic field (9.4 T).

Regulation of apoptosis is critical for cell survival during mild stress and for proper removal of damaged cells during severe stress including hyperthermia. Previous studies have shown that knockdown of the BH3-only protein Noxa prevents hyperthermia-induced Mcl-1 degradation and activation of apoptosis. Noxa is a pro-apoptotic BH3-only protein that is able to selectively bind to and disable anti-apoptotic Mcl-1. Phosphorylation of Noxa on serine-13 by the cyclin-dependent kinase CDK5 inhibits the apoptotic function of Noxa. In this study I investigated whether hyperthermia is able to induce apoptosis by preventing Noxa phosphorylation, due to reduced CDK5 activity, leading to activation of Noxa. I was able to demonstrate that both the phosphorylation status and solubility of CDK5 is reduced during hyperthermia. Furthermore, overexpression of a non-phosphorylatable Noxa (S13A) resulted in a significant decrease in cell viability and increase in caspase-3 activity compared to overexpression of wild-type Noxa at 37°C. However, I was unable to detect in vivo phosphorylation of Noxa serine-13 in lymphoid cells and therefore was unable to conclude whether or not hyperthermia affects the phosphorylation status of Noxa.

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