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Yes / Cancer-causing mutations often arise from inappropriate DNA repair, yet acute exposure to DNA damage is widely used to treat cancer. The challenge remains in how to specifically induce excessive DNA damage in cancer cells while minimizing the undesirable effects of genomic instability in noncancerous cells. One approach is the acute exposure to hyperthermia, which suppresses DNA repair and synergizes with radiotherapy and chemotherapy. An exception, however, is the protective effect of hyperthermia on topoisomerase targeting therapeutics. The molecular explanation for this conundrum remains unclear. Here, we show that hyperthermia suppresses the level of topoisomerase mediated single- and double-strand breaks induced by exposure to topoisomerase poisons. We further uncover that, hyperthermia suppresses hallmarks of genomic instability induced by topoisomerase targeting therapeutics by inhibiting nuclease activities, thereby channeling repair to error-free pathways driven by tyrosyl-DNA phosphodiesterases. These findings provide an explanation for the protective effect of hyperthermia from topoisomerase-induced DNA damage and may help to explain the inverse relationship between cancer incidence and temperature. They also pave the way for the use of controlled heat as a therapeutic adjunct to topoisomerase targeting therapeutics.

Yes / Magnetic Field Hyperthermia is a technique where tumours are treated through an increase in local temperature upon exposure to alternating magnetic fields (AMFs) that are mediated by magnetic nano-particles (MNPs). In an AMF, these particles heat-up and kill the cells. The relationship between an AMF and the heating-rate is complex, leading to confusion when comparing data for different MNP and AMF conditions. This work allows for the thermal-response to be monitored at multiple AMF amplitudes while keeping other parameters constant. An induction-heating coil was designed based on a Zero-Voltage-Zero-Current (ZVZC) resonant circuit. The coil operates at 93 kHz with a variable DC drive-voltage (12–30 V). NEC4 software was used to model the magnetic field distribution, and MNPs were synthesised by the coprecipitation method. The magnetic field was found to be uniform at the centre of the coil and ranged from 1 kAm−1 to 12 kAm−1, depending on the DC drive-voltage. The MNPs were found to have a specific absorption rate (SAR) of 1.37 Wg−1[Fe] and 6.13 Wg−1[Fe] at 93 kHz and 2.1 kAm−1 and 12.6 kAm−1, respectively. The measured SAR value was found to be directly proportional to the product of the frequency and field-strength (SARα f Ho). This leads to the recommendation that, when comparing data from various groups, the SAR value should be normalized following this relationship and not using the more common relationship based on the square of the field intensity (SARα f Ho2). / This work is partially supported by the UK Engineering and Physical Sciences Research Council (EPSRC) under grant EP/X039366/1, and HORIZON-MSCA-RISE ID: 101086492, Marie Skłodow-ska-Curie, Research and Innovation Staff Exchange (RISE), titled: FractuRe Orthopaedic Rehabilita-tion: Ubiquitous eHealth Solution (Robust).

Doctor of Philosophy / Department of Anatomy and Physiology / Deryl L. Troyer / Many cell types were known to have migratory properties towards tumors and different research groups have shown reliable results regarding cells as delivery vehicles of therapeutics for targeted cancer treatment. Present report discusses proof of concept for 1. Cell mediated delivery of Magnetic nanoparticles (MNPs) and targeted Magnetic hyperthermia (MHT) as a cancer treatment by using in vivo mouse cancer models, 2. Cells surface engineering with chimeric proteins for targeted cancer treatment by using in vitro models. 1. Tumor homing cells can carry MNPs specifically to the tumor site and tumor burden will decrease after alternating magnetic field (AMF) exposure. To test this hypothesis, first we loaded Fe/Fe3O4 bi-magnetic NPs into neural progenitor cells (NPCs), which were previously shown to migrate towards melanoma tumors. We observed that NPCs loaded with MNPs travel to subcutaneous melanoma tumors. After alternating magnetic field (AMF) exposure, the targeted delivery of MNPs by the NPCs resulted in a mild decrease in tumor size (Chapter-2). Monocytes/macrophages (Mo/Ma) are known to infiltrate tumor sites, and also have phagocytic activity which can increase their uptake of MNPs. To test Mo/Ma-mediated MHT we transplanted Mo/Ma loaded with MNPs into a mouse model of pancreatic peritoneal carcinomatosis. We observed that MNP-loaded Mo/Ma infiltrated pancreatic tumors and, after AMF treatment, significantly prolonged the lives of mice bearing disseminated intraperitoneal pancreatic tumors (Chapter-3). 2. Targeted cancer treatment could be achieved by engineering tumor homing cell surfaces with tumor proteases cleavable, cancer cell specific recombinant therapeutic proteins. To test this, Urokinase and Calpain (tumor specific proteases) cleavable; prostate cancer cell (CaP) specific (CaP1 targeting peptide); apoptosis inducible (Caspase3 V266ED3)- rCasp3V266ED3 chimeric protein was designed in silico. Hypothesized membrane anchored chimeric protein (rCasp3V266ED3, rMcherry red) plasmids were constructed. Membrane anchoring and activity of designed proteins were analyzed in RAW264.7 Mo/Ma and HEK293 cells in vitro. Further, Urokinase (uPA) mediated cleavage and release of rCasp3V266ED3 from engineered cells was tested (Chapter-4). Animal models for cancer therapy are invaluable for preclinical testing of potential cancer treatments. Final chapter of present report shows evidence for immune-deficient line of pigs as a model for human cancers (Chapter-5)

Presented in this thesis are the following theoretical investigations carried out on the non-invasive microwave hyperthermia of malignant tumours in the human body: Fundamental concepts of electromagnetic wave propagation through a biomass and its interaction with it, are discussed. Various types of applicators used for producing hyperthermia in a biomass, are also discussed. Propagation of a uniform plane electromagnetic wave through a human body is investigated for the general case of oblique incidence. Various models used for the human body have been discussed and the planar multilayer model has been chosen for this study. Reflection and transmission coefficients for both the parallel and perpendicular linear polarisations of the wave, have been determined. For normal incidence, power transfer ratio at the muscle has been defined and calculated at 433, 915 and 2450 MHz (ISM frequencies). Efects of skin thickness and also of fat thickness, on the power transfer ratio at muscle, have been studied. Effects of the thickness and dielectric constant of a bolus, and also of the dielectric constant of an initial layer, on the power transfer ratio, have been studied and their optimum values obtained at the ISM frequencies. For microwave hyperthermia, 915 MHz is recommended as the frequency of operation. Steady-state solution of the bioheat transfer equation has been obtained, assuming the biomass to be a semi-infinite homogeneous medium. Effects of various physical parameters on the temperature profile in the biomass, have been studied. Also studied is the effect of the surface temperature on the magnitude, location and the width of the temperature peak attained in the biomass. A method to determine the microwave power and the surface temperature required to produce a prescribed temperature profile in the biomass, has been developed. The transient-state solution of the bioheat transfer equation has been obtained to study the building up of the temperature profile. Procedures for the design of an open-ended rectangular metal waveguide applicator and for estimating the total microwave power requirement to produce hyperthermia in the human body, have been developed. Performance of the applicators employing linear as well as planar arrays of open-ended rectangular metal waveguide antennas, has also been studied. In order to reduce the overall physical size of the applicators, filling up of the feed waveguide with a high dielectric constant but low loss material is suggested. A simple method of obtaining the elements of the array by partitioning a large aperture by using metal walls has been adopted. Calculation of the total microwave power required by various applicators for producing hyperthermia at various depths in a biomas, have been made and a comparison of the performance of various applicators, has been presented.

Doctor of Philosophy / Department of Chemistry / Stefan Bossmann / My thesis is divided into two parts. The first part is focused on studies of N-heterocyclic carbene (NHC) palladium(IV) intermediates, which are involved in oxidative addition mediated C-C, and C-O bond formation processes as well as in C-Cl bond forming reactions via a reductive elimination process. Bis-NHC-Pd(II) complexes have been reported as effective catalysts to mediate direct conversion of methane into methanol. However, a H-D exchange study revealed that the bis-NHC-Pd(II) complexes are not the active species responsible for the C-H bond activation reaction. This unexpected result implies that the high oxidation state bis- NHC-Pd(IV) species may be the real catalyst! The oxidative addition of methyl iodide to the bis- NHC-Pd(II)-Me2 complex led to the successful observation of the formation of a transient trimethyl bis-NHC-Pd(IV) intermediate by both 1H-NMR and 13C-NMR spectroscopy. Different oxidants such as O2, PhI(OAc)2, PhI(OTFA)2 and Cl2 reacted with the bis-NHC-Pd(II)-Me2 complex, and competitive C-C and C-O bond formations, as well as C-C and C-Cl bond formations were observed. Dioxygen triggered C-C bond formation under dry condition and both C-C and C-O bond formation in the presence of H2O gave strong indications that the bis-NHCPd( II)-Me2 complex can be oxidized to a bis-NHC-Pd(IV) intermediate by dioxygen. The reaction between the hypervalent iodine regents PhI(OAc)2 and PhI(OTFA)2 and the bis-NHCPd( II)-Me2 complex gave only reductive elimination products. Therefore, this system can act as a model system, which is able to providing valuable information of the product forming (functionalization) step of the C-H bond activation system. The reaction between chlorine and the bis-NHC-Pd(II)-Me2 complex resulted in a relatively stable bis-NHC-Pd(IV)-Cl4 complex, which was characterized by 1H-NMR spectroscopy and mass spectroscopy. The structure of bis- NHC-Pd(IV)-Cl4 was unambiguously established by X-ray crystallography. The second part of this thesis describes the synthesis of functionalized bimagnetic core/shell iron/iron oxide nanoparticles for the treatment of cancer. Biocompatible dopamineoligoethylene glycol functionalized bimagnetic core/shell Fe/Fe3O4 nanoparticles were prepared via ligand exchange, and purified by repeated dispersion/magneto-precipitation cycles. A porphyrin (TCPP) has been tethered to the stealth nanoparticles to enhance their uptake by tumor cells and (neural) stem cells. The stealth nanoparticles have been delivered in a mouse model to tumor sites intravenously by using the EPR (enhanced permeation and retention) effect. Magnetic hyperthermia proved to be very effective against B16-F10 mouse melanomas in Charles River black mice. After hyperthermia, the nanoparticles have shown a significant effect on the growth of tumor (up to 78% growth inhibition).

Master of Science / Department of Electrical and Computer Engineering / Punit Prakash / Thermal therapies employing interstitial microwave applicators for hyperthermia or ablation are in clinical use for treatment of cancer and benign disease in various organs. However, treatment of targets in proximity to critical structures with currently available devices is risky due to unfocused deposition of energy into tissue. For successful treatment, complete thermal coverage of the tumor and margin of surrounding healthy tissue must be achieved, while precluding damage to critical structures. This thesis investigates two approaches to increase precision of microwave thermal therapy. Chapter 2 investigates a novel coaxial antenna design for microwave ablation (MWA) employing a hemi-cylinderical reflector to achieve a directional heating pattern. A proof of concept antenna with an S₁₁ of -29 dB at 2.45 GHz was used in ex vivo experiments to characterize the antennas’ heating pattern with varying input power and geometry of the reflector. Ablation zones up to 20 mm radially were observed in the forward direction, with minimal heating (less than 4 mm) behind the reflector. Chapter 3 investigates the use of magnetic nanoparticles (MNP) of varying size and geometry for enhancing microwave tissue heating. A conventional dipole, operating at 2.45 GHz and radiating 15 W, was inserted into a 20 mm radius sphere of distributed MNPs and heating measurements were taken 5 mm, 10 mm, and 15 mm radially away. A heating rate of 0.08°C/s was observed at 10 mm, an increase of 2-4 times that of the control measurement. These approaches provide strong potential for improving spatial control of tissue heating with interstitial and catheter-based microwave antennas.

Master of Science / Department of Anatomy and Physiology / Deryl L. Troyer / There is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy. The influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/Fe3O4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands. The magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe3O4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. There is a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure. These results indicate that intratumoral administration of surface-modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, intravenous administration of these MNPs followed by AMF exposure attenuates melanomas, indicating that adequate amounts of TCPP-labeled stealth Fe/Fe3O4 nanoparticles can accumulate in murine melanoma after systemic delivery to allow effective magnetic hyperthermic therapy in a rodent tumor mode.