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<p>Ionizing radiation is without a doubt an invaluable tool in diagnostic imaging as well as radiation therapy. With the growing number of medical and occupational exposures, together with challenges against the LNT model, low dose exposures and non-targeted effects have been subject to intensive research. Additionally, with the advances in the field of radiation therapy and longer life expectancy after the treatment, the risks associated with second malignancies following radiation therapy for various cancers has received a tremendous amount of attention. On the other hand, nicotine, as the addictive component of tobacco has been known for its adverse health effects and its relation to various types of cancers, accounting for one in 10 adult deaths worldwide. Both nicotine and low doses of radiation are amongst the stressors that widely affect the public. Surprisingly, the interactions between low-dose effects and nicotine exposure have not received the proper scientific attention. Our group has been involved in investigation of the non-targeted effects of radiation with a variety of endpoints. Different natural compounds and signalling molecules have also been studied in our lab for their possible role or contribution to bystander signalling. This research involves the study of the impact of nicotine on radiation-induced bystander effects and also radioadaptive responses. Different concentrations of nicotine were used to study the kinetics of the drug as well as any detrimental or modifying effects when used together with radiation. It was shown that nicotine has a protective effect on survival of the cells in certain concentrations that follows a biphasic model. Similar bimodal behaviour was observed with bystander effect. No adaptation to a challenge dose of radiation occurred as a result of incubation with varying concentrations of nicotine, nor was such an effect shown with a priming dose of radiation. The results of the present study suggest that nicotine has a complicated effect on the cells which can vary significantly depending on the concentrations used and also the duration of exposure. nAChRs may have an important role in the response of the bystander cells when nicotine is involved as the results showed a shift in the response of the receptors to nicotine. This thesis is aimed to shed light on the impact of nicotine and initiate more detailed investigations on pathways through which these effects are mediated.</p> / Master of Science (MSc)

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

Glioblastoma is the most commonly diagnosed central nervous system primary malignancy and it is considered a terminal diagnosis with few treatment options available. Glioblastoma tumors frequently develop treatment resistance due in part to their highly heterogenic nature. The heterogeneity of glioblastoma is partially attributed to the presence of glioma stem-like cells (GSC), which are highly invasive and resistant to chemotherapy and irradiation treatments. Signaling of the receptor tyrosine kinase MET is a known regulator of GSC. Glioblastoma patients have an increasingly poor prognosis that corresponds with increasing MET expression. Both GSC and MET are known to contribute to treatment resistance in glioblastoma and several MET alterations have been observed in glioblastoma. In these studies, we investigated MET alterations that are commonly found in glioblastoma. Using patient-derived xenograft (PDX) lines, the MET alterations were characterized and confirmed to be MET positive, MET amplified, or harbor a PTPRZ1-MET fusion. We also included a MET null glioblastoma PDX line. The PDX lines demonstrated markers for GSC potential with all showing neurosphere formation, the ability to initiate tumor growth in immune-compromised mice, and expression of GSC markers GFAP, Sox2, and nestin. The MET alterations were further examined by examining tyrosine kinase inhibitors' effect on viability and MET signaling. Oncogene addiction through MET amplification was found to have the best response to inhibition. The MET fusion bearing line demonstrated less sensitivity to inhibition than has been shown in other studies, indicating a need for further research into co-mutations that increase sensitivity to MET inhibition. We also investigated the efficacy of novel MET-targeting chimeric antigen receptor T cells (MET.CART cells). The MET.CART cells were able to specifically target and successfully kill MET-expressing glioblastoma cells. Together these results imply the need for more personalized treatment of glioblastoma based on the molecular biology of the tumor.

The field of biomedical sciences has been expanded through the introduction of a novel cohort of soft and intelligent microrobots that can be remotely operated and controlled through the use of external stimuli, such as ultrasound, magnetic fields, or electric fields, or internal stimuli, such as chemotaxis. The distinguishing factor of these microrobots lies in their propulsion system, which may encompass chemical, physical, or biohybrid mechanisms. Particularly, microrobots propelled by motile cells or microorganisms have found extensive usage because they combine the control/steerability and image-enhancement capabilities of the synthetic microstructures with the taxis and cell-interaction capabilities of the biological components. Spermatozoa (sperms), among other types of motile microorganisms and cells, are promising biological materials for building biohybrid microrobots because they are inherently designed to swim through complex fluids and organs, like those in the reproductive system, without triggering negative immune responses. Sperms are suitable for a variety of gynecological healthcare applications due to their drug encapsulating capability and high drug-carrying stability, in addition to their natural role of fertilization. One objective of this project is to help sperms reach the site of fertilization in vivo where the sperm count is low (20 million sperm per mL), a condition known as oligospermia. In order to reach this goal, we are developing alternative strategies for transporting a significant number of sperms, as well as improving the functionality of sperm-hybrid microcarriers. Here, we use a thermoresponsive hydrogel made of poly(N-isopropylacrylamide) (PNIPAM) and a non-stimuli-responsive polymer (IPS photoresist) to create four dimensional (4D)-printed sperm-hybrid microcarriers via two-photon polymerization (TPP). We present a multifunctional microcarrier that can: i) transport and deliver multiple motile sperms to increase the likelihood of fertilization, ii) capacitate/hyperactivate the sperms in situ through the local release of heparin, and iii) assist the degradation of the hyaluronic acid (HA), present in extracellular matrix (ECM) of oocyte-cumulus surrounded the Egg. HA degradation occurs through the local action of hyaluronidase-loaded polymersomes (HYAL-Psomes) that have been immobilized on the microcarrier's surface. Dual ultrasonic (US)/photoacoustic (PA) imaging technology can also be used to visualize a swarm of microcarriers, making them ideal candidates for upcoming in vivo applications. In addition, as a second objective, we demonstrate that similar sperm-hybrid microcarriers can be utilized to deliver targeted enzymes and medication for the treatment of gynecological cancer. As proof of concept, we show that combined therapy using enzymes and anti-cancer drugs is an appealing strategy for disrupting the tumor tissue microenvironment and inducing cell apoptosis, thereby offering a more effective cancer therapy. To achieve this, we functionalize the microcarriers with polymersomes loaded with enzymes (such as hyaluronidase and collagenase) and anti-cancer drugs (such as curcumin), respectively, and demonstrate their cargo-release capability, enzyme function, and therapeutic effect for targeting cervical cancer cells in vitro.:Abstract iv 1 Introduction 1 1.1 Motivation 1 1.2 Objectives 3 1.3 Structure of this dissertation 4 2 Background 5 2.1 Introduction on additive manufacturing technology 5 2.2 Direct laser writing (DLW) based on two-photon polymerization 6 2.2.1 Writing principles of two-photon lithography 8 2.2.2 Available materials for two-photon lithography 9 2.2.3 Engineering (Preprogrammed designs) 12 2.3 4D Lithography 13 2.3.1 Biodegradable microrobot 13 2.3.2 Stimuli-responsive micromotors 15 2.3.3 Other 4D-printing approaches 17 2.4 Motion at the microscale (Micromotility) 21 2.4.1 Physical propelled micromotors 23 2.4.2 Chemical propelled micromotors 32 2.4.3 Biohybrid micromotors 34 2.5 Other two-photon polymerized microrobots and their biomedical applications 35 2.5.1 Functionalized carriers 36 2.5.2 Multiple-cell carrying scaffolds 38 2.5.3 Single particle and cell transporters 39 2.6 Comparison of 3D and 4D-lithography with other fabrication methods 42 3 Materials and methods 44 3.1 Synthesis and fabrication 44 3.1.1 Synthesis of PNIPAM 44 3.1.2 Fabrication of microcarrier 44 3.1.3 Preparation of sperm medium and sperm solution 45 3.1.4 Preparation and composition of different body fluids 45 3.1.5 Fluidics channels 46 3.1.6 In situ preparation of microcarriers and sperms 46 3.1.7 Loading of microcarriers with heparin 46 3.1.8 Synthesis of block copolymers (BCPs) 47 3.1.9 Fabrication of Empty-Psomes A and D 48 3.1.10 Preparation of Curcumin complex CU(βCD)2 and calibration curve 49 3.1.11 Fabrication of cargo-loaded Psomes with enzymes and antitumoral drug 50 3.2 Characterization 51 3.2.1 MTS-Assay 51 3.2.2 Toluidine blue assay 52 3.2.3 Characterization of Empty-Psomes A and D: pH cycles and pH titration by dynamic light scattering (DLS) 53 3.2.4 Characterization of cargo-loaded Psomes with enzymes and antitumoral drug 54 3.2.5 Loading efficiency of HYAL-Psomes 55 3.2.6 Loading efficiency of MMPsomes 56 3.2.7 Loading efficiency, stability and release study of CU(βCD)2-Psomes 57 3.2.8 Size and polydispersity analysis of cargo-loaded Psomes in different simulated body fluids by DLS 58 3.2.9 Conformation and stability study of cargo-loaded Psomes in different simulated body fluids by asymmetric flow field flow fractionation (AF4) 59 3.2.10 Immobilization of the cargo-loaded Psomes on the surfaces 61 3.2.11 Enzymatic assay of HYAL for enzyme activity measurement 62 3.2.12 Enzymes assay in different simulated body fluids 64 3.2.13 Stability study of RhB-HYAL-Psomes in different pH 65 3.2.14 Calculation of the magnetic field flux of an external hand-held magnet 66 3.3 Temperature actuation and imaging 67 3.3.1 Temperature actuation test of PNIPAM and video recording 67 3.3.2 Hybrid ultrasound (US) and photoacoustic (PA) Imaging 67 3.4 Other useful information 68 3.4.1 pH and temperature through the female reproductive tract 68 3.4.2 Calculation of the light-to-heat conversion during imaging process 69 4 Multifunctional 4D-printed sperm-hybrid microcarriers for assisted reproduction 72 4.1 Background 72 4.2 Concept and fabrication of the 4D-printed microcarriers 74 4.3 Sperm coupling and geometrical optimization of microcarrier 77 4.4 Characterization of the 4D-printed streamlined microcarriers 78 4.5 Microcarrier loaded with heparin for in situ sperm capacitation 82 4.6 Microcarriers decorated with HYAL-Psomes for in situ degradation of the HA-cumulus complex 86 4.6.1 Immobilization of HYAL-Psomes on the microcarrier’s surface 89 4.6.2 Qualitative study of cumulus cell removal 90 4.7 Sperm-microcarrier motion performance in oviduct-mimicking fluids 91 4.7.1 Capture, transport, and release of sperms 92 4.7.2 Sperm-microcarrier motion performance on ex vivo oviduct tissue 93 4.8 Tracking of a swarm of microcarriers with a dual ultrasound (US) and photoacoustic (PA) imaging system 95 4.9 Summary 96 5 Polymersomes-decorated micromotors with multiple cargos for gynecological cancer therapy 98 5.1 Background 98 5.2 Characterization and size quantification of Psomes before and after loading of cargoes by DLS, and Cryo-TEM 103 5.3 Characterization and size quantification of cargo-loaded Psomes by DLS, and Cryo-TEM in different simulated bodily fluids 104 5.4 Immobilization and characterization of cargo-loaded Psomes on the microcarrier’s surface 106 5.5 Immobilization and characterization of dual cargo-loaded Psomes on the microcarrier’s surface 108 5.6 Investigation of ECM degradation and antitumoral effect of cargo-loaded Psomes 110 5.7 Magnetic and bio-hybrid guidance of microcarriers toward targeted cargo delivery 115 5.8 Summary 117 6 Conclusion and Outlook 119 6.1 Achievements 119 6.2 Outlook 121 Bibliography I List of Figures and Tables XXI Acknowledgements and funding XXIV Scientific publications and contributions XXVI Curriculum Vitae XXVII

This study examines the business model of Meta called “Pay or OK”, a subscription modelthat offers users the choice to either consent to the use of Meta’s services and be subjected totargeted advertising or to use the service without personalized advertising for a fee. In the eraof digital transformation, where data has become a significant competitive advantage forlarge online platforms, the introduction of regulations like GDPR and DMA has beeninstrumental in protecting personal integrity and ensuring fair competition. This study utilizesqualitative and EU legal methods to analyze the “Pay or OK” business model from a legalperspective and also analyze if data could create competitive advantage for Meta,incorporating theories such as the resource-based view and the VRIO framework. The resultsof this study bring to the forefront the intriguing and contrasting opinions on the value ofpersonal data and the profound impact of regulations on the business landscape.

This research explores the relationship between school shooter typologies, warning signs, and targeted school violence. The study's independent variables are school shooter typologies and warning signs, while the dependent variable is targeted school violence. The research wants to investigate whether a relationship exists between these variables. The null hypothesis states no relationship exists, while the alternative hypothesis suggests that a relationship exists. The research framework is based on Bronfenbrenner's Ecological System Theory, which helps understand how the environment and social interaction influence a child's development. The theory was influenced by Vygotsky's sociocultural theory—which focuses on the roles of human interactions and culturally structured activities in cognitive functioning that affect psychological growth, and Lewin's behaviorism theory—which proposed that behavior results from the individual and the environment. The findings show a strong, positive relationship, high correlation, and statistical significance difference between school shooter typologies, warning signs, and targeted school violence. The alternate hypothesis was accepted based on the findings, indicating that the relationship between these variables is not due to a coincidence or chance alone. These findings can offer valuable insights to policymakers in making informed decisions on allocating school safety resources to prevent targeted school violence.

No / The identification of an agent that is selectively activated by a cytochrome P450 (CYP) has the potential for tissue specific dose intensification as a means of significantly improving its therapeutic value. Towards this goal, we disclose evidence for the pathway of activation of a duocarmycin analogue, ICT2700, which targets CYP1A1 for biological activity.

Lay Abstract / Prostate cancer (PrCa) is a leading cause of cancer-related death in men worldwide. Standard treatments for unresected PrCa include radiation therapy, androgen deprivation or anti-androgen therapies. However, many patients develop resistance to these therapies indicating a need for a new treatment that can enhance their therapeutic efficacy. Our studies show a promising new strategy to achieve this task using canagliflozin, an FDA-approved medication currently used to treat diabetes, kidney and heart failure. We found that canagliflozin interrupts cellular energy production, inhibits biosynthetic pathways and improves the efficacy of radiotherapy in radiation naïve and radio-resistant human PrCa models. In addition, we found that canagliflozin has promising anti-androgen activity by reducing the protein levels and the activity of androgen receptor (AR) in PrCa cells and tumors both alone and in combination with commercially available anti-androgens. These promising findings suggest that canagliflozin could be used in combination with current standard-of-care therapies for the treatment of PrCa. Given that canagliflozin is an approved medication, tolerated well by diabetic and non-diabatic individuals, the work presented here generates a basis for rapid investigation of this new treatment in early phase clinical trials. / Thesis / Doctor of Philosophy (PhD)