Heat stress (HS) is one of the consequential important problems facing the swine industry. The negative effects of HS include reduced growth performance, reproductive efficiency, and carcass quality as well as increased morbidity and mortality. Although, the swine industry has developed several abatement strategies (i.e., fans, cooling pads, sprinklers, etc.), these approaches may be ineffective in the future as global temperatures continue to rise and the frequency of more severe heat waves increases in regions where animal agriculture is prevalent. These extreme heat events put pigs (especially those approaching market weight) at risk for acute hyperthermia that can lead to death unless body temperature is rapidly returned to euthermia and thermoregulatory function is restored.Therefore, evaluating mitigation strategies to promote recovery from acute hyperthermia is of utmost importance for improving pigs’ health and well-being and ensuring profitability and food security. In four experiments, the existence of microclimates in grow-finish barns during late summer was ascertained and a rapid cooling technique using cold water dousing and feed removal to promote recovery from acute hyperthermia in pigs was evaluated. In the first study, it was determined that microclimates exist in grow-finish barns and that pigs raised in pens that were not located directly below air inlets and ventilation fans had greater body temperature and reduced feed efficiency despite similarities in the in-barn ambient temperature and relative humidity. These data exemplifythe importance of adequate ventilation systems in swine barns and the impact of microclimates on pigs’ health and productivity during warm summer months. In the second study, grow-finish pigs that did not have feed access were exposed to acute HS and then rapidly or gradually cooled. Following the acute HS and recovery phase, all pigs were maintained under thermoneutral conditions and then euthanized over three days to determine the temporal effects of the cooling treatment on body temperature and intestinal integrity. The results showed that rapid cooling following acute hyperthermia in pigswas effective in returning body temperature to euthermia more rapidly compared to gradual cooling and rapid cooling prevented further intestinal damage. Based on these results, it was hypothesized that feed removal may have played a role in the effectiveness of rapid cooling. Therefore, a third experiment was conducted in which grow-finish pigs with or without access to feed were exposed to an acute HS challenge and then rapidly cooled. This study concluded that feed access was a determinant factor in the cooling outcome, as the gastrointestinal temperature returned to euthermia during the rapid cooling period more rapidly when feed was removed. Finally, a fourth study was conducted to evaluate the effects of feed removal in the absence of rapid cooling on the systemic inflammatory response and short-term growth performance of grow-finish pigs. However, it was determined that feed removal alone did not reduce the inflammatory response as expected. Overall, these studies demonstrate the risk forgrow-finish pigs during summer heat events and the potential use of rapid cooling in combination with feed removal for promoting recovery from acute hyperthermia in pigs.

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Focused ultrasound (FUS) is a less-invasive medical technique with the potential to improve the treatment outcome of many diseases by utilizing acoustic transducers to generate and concentrate the multiple intersecting ultrasonic waves on a targeted site in the body. The bio-effects induced by FUS are mostly classified into thermal and mechanical effects (mainly focus on cavitation effect). Cavitation is capable of disrupting tumor vasculature and cell membranes. Numerous studies reported that cavitation-induced sonoporation could provoke multiple anti-proliferative effects on cancer cells, including cell-cycle arrest, cell apoptosis, and clonogenicity suppression. Therefore, the combination of FUS-induced cavitation and other treatment modalities like radiation therapy is of great interest, but research in this field is inadequate. A special high-throughput FUS system was used for cancer cell treatment with a customized 1.467 MHz single focused transducer. Characterization of acoustic behavior of gas-filled cavities was performed via a fiber-optic hydrophone (FOH) system and chemical terephthalic acid method helped to define the acoustic parameters, which could lead to occurrence of cavitation at the bottom of 96-well cell culture plates where cancer cells were located. Cavitation occurs at and above the acoustic intensity of 344 W/ cm2 for the 1.467 MHz transducer. The short- and long-term effects of FUS-induced cavitation and adjuvant effects to radiation therapy, standard hyperthermia and testosterone treatment (only for prostate cancer) were investigated comprehensively at the cellular and molecular levels in human prostate cancer (PC-3 and LNCap), glioblastoma (T98G) and head and neck (FaDu) cells in vitro. The long-term additive effects of short FUS shots (with or without cavitation) to radiation therapy (RT) or hyperthermia (HT) were displayed by significantly reduced clonogenic survival in PC-3, T98G and FaDu cells compared to single treatments. The combination treatment of short FUS with cavitation (FUS-Cav) and RT led to a comparable radio-sensitization effect to HT at 45 °C for 30 min and showed a significant reduction in treatment duration, especially for PC-3 cells. The short-term additive effects of short FUS shots to RT or HT are manifested in reducing the potential of cells to invade and decreased metabolic activity. The induction of sonoporation by FUS-Cav was supposed to be the mechanism of cancer cell sensitization to other therapies at the cellular level. The dramatic decline of 5α-reductase type III (SRD5A3) level induced by combination treatment with FUS-Cav and HT is presumed as the underlying mechanism of additive effects of FUS-Cav to HT at the molecular level. Besides, testosterone solutions with normal physiological levels were discovered to inhibit the long-term metabolic activity of androgen-dependent prostate cancer cells LNCap in vitro, while short FUS shots displayed a long-term additive effect to the testosterone treatment. The presented multilevel study demonstrates that short FUS shots using FUS-induced cavitation carry the potential to sensitize cancer cells to other cancer treatment modalities precisely and less-invasively, providing a promising adjuvant therapy to cancer treatments in the future.:1 Abbreviations 2 Summary 3 Introduction 4 Medical and technical background 4.1 The biological basis of prostate cancer treatment 4.1.1 Androgen receptor: an essential signaling pathway for progression of prostate cancer 4.1.2 5α-reductase: a promising therapeutic target for prostate cancer therapy 4.1.3 Testosterone: duality effects in prostate cancer development 4.2 Advantages and disadvantages of current clinical treatments of prostate cancer 4.3 Basics of focused ultrasound (FUS) 4.3.1 Medical application of FUS-induced thermal effects 4.3.1.1 High-intensity focused ultrasound (HIFU) induced thermal ablation 4.3.1.2 Hyperthermia: an alternative heating strategy to sensitize cancer cells for radiation therapy and chemotherapy 4.3.1.3 FUS-induced hyperthermia triggered drug delivery with thermo-sensitive drug carriers 4.3.2 Medical application of FUS-induced mechanical/cavitation effects 4.3.2.1 Cell sonoporation for drug delivery 4.3.2.2 Sonoporation induced anti-proliferative effects for cancer cells 4.3.2.3 Histotripsy 4.3.2.4 Anti-vascular and anti-metastatic effects 4.3.3 The state of art of cavitation detection in medical application 4.3.3.1 Sonoluminescence and sonochemistry 4.3.3.2 Passive cavitation detection 4.3.3.3 Active cavitation detection 4.3.3.4 High-speed sequential photography of cavitation dynamics 4.3.3.5 Laser scattering technique 4.3.3.6 Synchrotron X-ray imaging technique 4.3.3.7 MRI techniques 5 Aims of the thesis 6 Materials and methods 6.1 Materials 6.1.1 Devices 6.1.2 Chemicals and reagents 6.1.3 Consumables 6.1.4 Human cancer cell lines 6.2 Methods 6.2.1 Composition of the FUS system for in vitro treatment of cells 6.2.2 Physical characterization of the in vitro FUS system 6.2.2.1 Setup of fiber-optic hydrophone system to characterize the FUS apparatus 6.2.2.2 Data analysis in MATLAB 6.2.3 Cavitation measurement with FOH system 6.2.4 Chemical cavitation measurement with terephthalic acid (TA) 6.2.5 Culture of human cancer cell lines 6.2.6 FUS treatment of cancer cells 6.2.7 Water bath hyperthermia treatment 6.2.8 Radiation therapy in vitro 6.2.9 The protocol of FUS\FUS-Cav combined with RT or HT 6.2.10 Evaluation of cell ability to reproduce with clonogenic assay 6.2.11 Measurement of cellular metabolic activity with WST-1 assay 6.2.12 Evaluation of cell invasion ability with Transwell® assay 6.2.13 Detection of sonoporation by cell staining with propidium iodide (PI) 6.2.14 Detection of SRD5A as a therapeutic target for prostate cancer with immunofluorescence microscopy 6.2.15 Quantification for the reduction of SRD5A proteins with flow cytometry 6.2.16 Testosterone treatment 6.2.17 FUS/FUS-Cav combined with testosterone treatment 7 Results 7.1 Physical characterization of the in vitro FUS system 7.2 Cavitation occurs at a certain level of acoustic intensity 7.2.1 Characteristics of ultrasonic spectrograms 7.2.2 Cavitation dose depends on the acoustic intensity 7.3 FUS/FUS-Cav supports RT to reduce the long-term clonogenic survival of cancer cells 7.4 FUS/FUS-Cav increases the effects of HT by reducing the long-term clonogenic survival of cancer cells 7.5 FUS/FUS-Cav enhances the suppressive effects of RT in short-term cell potential to invade and metabolic activity of prostate cancer cells 7.6 FUS/FUS-Cav supports HT to diminish the short-term cell potential to invade and metabolic activity of prostate cancer cells 7.7 FUS-Cav treatment immediately induces sonoporation effects in PC-3 and FaDu cells 7.8 FUS/FUS-Cav enhances the effects of HT by inhibiting the SRD5A protein level in prostate cancer cell lines 7.9 FUS-Cav enhances the effects of the testosterone treatment by reducing the long-term cell metabolic activity of androgen-dependent prostate cancer cell line 8 Discussion 8.1 Cavitation measurement in a defined 96-well plate by PCD technique and sonochemistry method 8.2 Short-term and long-term additive effects of FUS-Cav to RT or HT 8.3 Inhibitory effects of FUS-cav in the potential of prostate cancer cells to invade 8.4 The reduction of the long-term metabolic activity of androgen-dependent prostate cancer cells by the combination treatment of FUS-Cav and testosterone 9 Conclusion 10 References 11 Appendix 11.1 Erklärung über die eigenständige Abfassung der Arbeit 11.2 List of figures 11.3 List of tables 11.4 Curriculum vitae 11.5 Acknowledgments

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In this dissertation a study of enhanced hyperthermia for cancer treatment through the use of magnetic nanoparticles is presented. Hyperthermia has been in use for many years, as a potential alternative method in cancer treatment, and high frequency microwave radiation has been used successfully to raise the tumor temperature to around 42°C in superficial tumors without causing damage to surrounding healthy tissues. Magnetic fluid hyperthermia involves the use of magnetic nanoparticles injected into the tumor before exposure to microwave radiation. The magnetic energy in the nanoparticles is converted into heat allowing for a more rapid rise of temperature in the tumor to the desired level. In addition, the nanoparticles allow the electromagnetic absorption to be focused in the tumor and can be used to treat deep tumors in organs, such as the liver. Iron oxide magnetic nanoparticles were considered for this study as they are non-toxic and bio-compatible. For the case of breast cancer, the values for the temperature and specific absorption rate (SAR) in the tumor and in the healthy tissue were obtained through simulations and validated by measurement done on phantom models. Various characteristics of the nanoparticles such as radius, magnetic susceptibility and concentration were considered. In order to take the effect of the blood flow, which causes cooling and helps maintain the body temperature, various blood perfusion rates for a tumor in the liver were studied. A human male model in SEMCAD X, in which blood flow can be adjusted, was used for simulations. The tumor was injected with the nanoparticles and the change in temperature upon exposure to electromagnetic radiation was observed. The simulated results were compared with measured results on a liver phantom model in which saline solution was used to model blood flow. There was good agreement between the measured and simulated results.

Steady advancements in diagnostics over the past century have propelled the world of medicine into the more advanced era of preventative medicine, an era with a resoundingly clear message: early detection can save lives. For patients who suffer from either pancreatic cancer or malignant hyperthermia susceptibility, early or preoperative diagnosis, respectively can save lives and minimize morbidity and mortality, in addition to offering cost-savings to hospitals and healthcare systems. Fortunately, significant progress have been made in the fields of metabolomics and biomarker identification. Given the benefits carried by serum biomarkers as targets of screening and diagnostic tool development, we applied functional nucleic acid technology and in vitro selection directly in whole human serum to search for disease-specific biomarkers and associated detection probes without a priori knowledge of the biomarkers pursued. This endeavour simultaneously serves as a proof-of-concept study to establish whether in vitro selection can be successfully performed in human serum. We specifically focused on the derivation of RNA-cleaving DNAzymes (RCD) through in vitro selection, or SELEX (systemic evolution of ligands through exponential exposure). DNAzymes constructed with a fluorogenic signalling molecule were incubated with human serum with the goal of identification of a functional nucleic acid probe capable of detecting the presence of a disease-specific biomarker. Two independent protocols have been designed and executed for the identification of DNAzyme sequences capable of detecting pancreatic cancer and malignant hyperthermia susceptibility, respectively. The first exploration was performed in serum obtained from cancer patients, with the goal of identifying DNAzymes capable of distinguishing pancreatic cancer from other cancer types. To do so, we employed in vitro selection, Next-Generation Sequencing, and bioinformatic analysis. We successfully demonstrated the feasibility of performing in vitro selection with DNAzymes in human serum, evidenced by distinct round-to-round enrichment of a DNA library towards the identification of DNAzymes capable of detecting pancreatic cancer. Additionally, we isolated two DNAzymes capable of distinguishing pancreatic cancer serum from healthy patient serum in fresh collected serum samples. Based on the positive results gathered in the pancreatic cancer in vitro selection project, we subsequently endeavoured to replicate the demonstrated feasibility of performing in vitro selection in human serum. By selecting malignant hyperthermia as the pathology investigated, we simultaneously sought to diversify the scope of DNAzyme detection by establishing whether successful DNAzyme selection can be achieved in a non-acute disease state. Thus, the second exploration was performed in serum obtained from patients who underwent evaluation for malignant hyperthermia susceptibility using the gold-standard caffeine-halothane contracture test. The goal of this project rested on the identification of DNAzymes capable of distinguishing malignant hyperthermia susceptibility in serum and approximating the performance of the gold standard test. We successfully isolated four DNAzyme candidates which demonstrated clinically relevant thresholds of sensitivity and specificity following thorough sensitivity and specificity analysis. In doing so, we once again demonstrated the ability to perform in vitro selection in human serum. Given the complexity of molecular interactions observed over the course of two in vitro selection protocols in human serum, it became clear that distinguishing meaningful target-mediated interactions from non-specific interactions would require advanced bioinformatic analysis. Consequently, using principles of computational biology, we performed a deep exploration of Next-Generation Sequencing results obtained from sequencing our recovered DNA libraries to extract additional data that would inform on the next required steps required to identify a DNAzyme specific for the pathology pursued. In doing so, we identified a two-step method to evaluate the progress of the in vitro selection protocol undertaken, and offered a systematic approach for choosing candidate sequences to undergo further testing based on promising performance in silico. Using this approach, we successfully identified a DNAzyme sequence capable of acting as a general cancer detection probe, with promising potential for diagnostic application. Ultimately, this thesis serves as a feasibility study of a novel approach to both in vitro selection and biomarker identification technique by combining the latest nanotechnology techniques with clinical data and real patient serum samples, and advanced computational biology tools. Despite the inability to identify a highly sensitive and specific DNAzyme capable of advancing towards biosensor construction, several important strides and lessons have been acknowledged, establishing the feasibility of performing in vitro selection in human serum, and outlining strategies for addressing and anticipating challenges with this technique. The hope is for this work to inspire and inform future efforts to apply functional nucleic acid technology to solve current gaps in both the diagnostic and therapeutic branches of medicine, and with the help of computational biology continue to bridge the gap between basic science and clinical medicine. / Dissertation / Doctor of Philosophy (PhD)

Focused ultrasound (FUS) has become an important non-invasive therapy for solid tumor ablation via thermal effects. The cavitation effect induced by FUS is thereby avoided but applied for lithotripsy, support drug delivery and the induction of blood vessel destruction for cancer therapy. In this study, head and neck cancer (FaDu), glioblastoma (T98G), and prostate cancer (PC-3) cells were exposed to FUS by using an in vitro FUS system followed by single-dose X-ray radiation therapy (RT) or water bath hyperthermia (HT). Sensitization effects of short FUS shots with cavitation (FUS-Cav) or without cavitation (FUS) to RT or HT (45 °C, 30 min) were evaluated. FUS-Cav significantly increases the sensitivity of cancer cells to RT and HT by reducing long-term clonogenic survival, short-term cell metabolic activity, cell invasion, and induction of sonoporation. Our results demonstrated that short FUS treatment with cavitation has good potential to sensitize cancer cells to RT and HT non-invasively.