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Retargeting AAV Vectors via Cell-Specific Receptor Binding
Full text linkIn recent years, the adeno-associated virus (AAV) has developed as the most widely-used vector for clinical gene therapy and basic biology research. AAV capsid has been extensively modified via different approaches to create the vectors with desired transduction efficiency and specificity in the target tissues. Nowadays, the majority of new capsids are developed from directed evolution in animal models because it does not require preliminary knowledge of the interaction between AAVs and target cells. However, in recent years, studies in different animal models have revealed that, variants selected in animals often fail to translate across species. One of the main reasons is the difference of receptors across species. Therefore, to develop AAV capsids that can be used in human for gene therapy, it is important to know the receptors that mediate the transduction.
In the study, I aimed to rationally design AAV vectors to transduce target cells by binding to cell-specific receptors. I worked on two major projects: (1) targeting to hACE2-expressing cells; and (2) penetration of the blood-brain-barrier (BBB) followed by targeting to glioblastoma (GBM). To achieve these goals, I displayed already known receptor-binding ligands (peptides and proteins) on the AAV capsid with three methods, namely, genetic insertion into the AAV capsid protein, chemical/covalent conjugation to the assembled AAV capsid, and non-covalent binding to the assembled AAV capsid. Here, I developed a site-specific chemical conjugation method, which allowed chemical conjugation of any kind of ligands on the desired site by introducing a cysteine residue. Furthermore, I adjusted the number of ligands displayed on the AAV capsid by producing mosaic capsids composed of wild-type VPs and ligand-displaying VPs. Thirdly, I displayed two ligands on the same capsid by producing mosaic capsids composed of two ligand-displaying subunits.
In the hACE2-targeting project, display of receptor binding domain of SARS-CoV-2 on the AAV capsid via non-covalent binding was successful and achieved the desired specific transduction of hACE2-expressing cells in vitro. In the BBB-penetration and GBM-targeting project, I identified two variants, AAV9-V4-FAL and AAV9-V8-FAL, displaying the EGFR and EGFRvIII binding peptide FALGEA, which transduced GBM efficiently in U87-transplanted nsg mice after intravenous administration and which were depleted from the major off-target tissue liver. Meanwhile, from the analysis of off-target tissues, I identified another variant, AAV9-V8-RTD, displaying the integrin αvβ6/αvβ8 binding peptide RTDLDSLRT, which transduced heart and muscle with high efficiency
Development of fully-human macrophage-modulatory antibodies for cancer immunotherapy
Full text linkTumor-associated macrophages (TAMs) contribute to tumor progression and foster the immunosuppressive tumor microenvironment. Several immunotherapeutic approaches to target TAMs are being developed, including the blockade of monocyte recruitment to the tumor site in order to reduce the number of pro-tumorigenic TAMs. Chemokine (C-C motif) ligand 2 (CCL2) and semaphorin 3A (Sema3A) secreted by tumor cells have been found to play crucial roles in the attraction of monocytes via the respective receptors C-C chemokine receptor type 2 (CCR2) and neuropilin 1 (NRP1) expressed on monocytes.
In this project, we selected fully human CCL2- and NRP1-targeting antibody fragments by means of phage display and further characterized them in the immunoglobulin 1 (IgG1) format. We demonstrated antigen specificity by ELISA, surface plasmon resonance spectroscopy and flow cytometry. Since we could not show blocking of the NRP1 – Sema3A interaction by the NRP1-specifc antibodies, these antibodies were not developed further. In contrast, we observed blocking of the CCL2 – CCR2 interaction by flow cytometry and confirmed the blockade of intracellular CCR2-dependent signaling mediated by our lead candidate anti-CCL2 antibody. Moreover, we demonstrated reduced CCL2-dependent migration of monocytes in the presence of the CCL2-specific antibody.
In addition, since recent reports showed that systemic administration of anti-CCL2 antibodies led to increased CCL2 production, we aimed to develop a system that allows for local and intratumoral delivery of the CCL2-neutralizing antibody fragment. To achieve this, we designed chimeric antigen receptor (CAR) constructs that enable simultaneous expression of the CAR and soluble secretion of our anti-CCL2 lead candidate. We demonstrated cell-surface expression of the CAR molecule and functionality of the CAR T cells. However, although simultaneous expression of the CAR and secretion of anti-CCL2 scFv was successfully proved for transfected HEK293T cells, we failed to detect scFv secretion from primary human T cells transduced with the same constructs. We assume that the amount of secreted scFv from T cells was below the detection limit of our assays. Thus, future work is warranted that shows that CAR T cells are able to produce sufficient amounts of anti-CCL2 scFv within the tumor and that this way of scFv delivery is superior to systemic administration routes
The contribution of transcription factors to chromatin accessibility at single molecule resolution
Full text linkMammalian genomes evolved to be very large and complex entities. The ca. 20,000 genes there encoded are precisely regulated in time, space and dosage to instruct for the development and the environmental adaptation of an organism. Gene expression, therefore, invokes a densely interconnected network of regulatory mechanisms in which sequence-specific transcription factors (TFs) occupy a central position. TFs characteristically bind to short DNA recognition motifs enriched at cis-regulatory elements (CREs), such as enhancers and promoters. Thereon, they tune gene expression by recruiting co-factors such as the basal transcription machinery and chromatin modification enzymes. In eukaryotes, the nucleosomes making up chromatin pose a physical barrier for the binding of TFs to DNA.
Understanding how TFs overcome this barrier at CREs is key to disentangle mammalian gene regulation. For example, certain TFs, pioneers, have the unique ability to establish accessibility de novo at inactive CREs. Whether they are equally pivotal for the maintenance of chromatin accessibility at active CREs is not clear. It is also not clear how important is the combinatorial assembly of multiple TFs and how TF- and CRE-specific this competition against nucleosomes is. Finally, it is to be determined whether CREs can function modularly or not.
In this project, I quantified TF-nucleosome competition in-vivo at CREs across the mouse genome. I did so at the single molecule level, at high coverage and near-nucleotide resolution using SMF, single molecule footprinting. SMF uses methylation footprinting to measure the frequency of CRE accessibility in a cell population, a proxy for the CRE- and TF-specific rate at which nucleosomes are outcompeted. Through its near-nucleotide resolution SMF captures footprints for TFs and nucleosomes, a leeway to investigate their interfacing.
To capitalize on this resolution, I developed two computational tools. The first, SingleMoleculeFootprinting, for the robust single molecule methylation calling, is currently distributed on Bioconductor. The second, FootprintCharter, currently under development, is an unsupervised tool for mapping and quantifying TF and nucleosome footprints.
To isolate the contribution of individual TF instances to accessibility at CREs, I quantified the effect of perturbing their binding across the mammalian genome. To that end, I exploited the natural genetic variation among different mouse species crossed into F1 lines. Such sequence variation often hinders the binding of one or few TFs at CREs and, therefore, their ability to outcompete nucleosomes. This highlights their effective contribution to CRE accessibility.
Among others, I observed a large degree of heterogeneity of TF-nucleosome competition across the mouse genome, with transcriptional enhancers showing the highest degree of variability. TF-nucleosome competition appears to be TF-specific, with Ctcf, Rest, Banp, Nrf1 and Nfya being most frequently successful. Pioneer TFs such as Klf4, Oct4 and Sox2 display a lesser individual contribution but assemble in context-dependent ways to outcompete nucleosomes. Nevertheless, most TF instances at CREs appear disposable for the maintenance of chromatin accessibility, highlighting pervasive CRE robustness. Finally, given how they react to perturbations, CREs seem to function in an all-or-nothing fashion rather than modularly.
With the work detailed in this dissertation, I shed some light on the contribution of transcription factors to chromatin accessibility at CREs. This work feeds into disentangling the hugely complex and vastly uncharted network of interactions that regulate eukaryotic gene expression
Navigating Barriers in Taiwanese Museums
Full text linkPeople with disabilities have often been framed as a burden to society and tend to have more difficulties to participate in society than people without disabilities. To this day they fight for equal rights. Reported cases of disabilities make up around 5% of Taiwan’s population. The amount of people with disabilities is increasing incrementally, which is partly due to an aging society. How Taiwan navigates accessibility issues can be analyzed by a look at how legislation and specifically cultural institutions have adapted to the needs of people with disabilities. The social responsibility of museums as cultural institutions to be inclusive has been widely discussed. To cement that fact the International Council of Museums (ICOM) has revised the definition of museum in 2022. Improvements in museum’s accessibility services to people with disabilities indicate its importance. The main focus of this research will be an analysis of accessibility of national museums in Taiwan. Accessibility is divided into architectural, informational and emotional accessibility. Architectural accessibility is analyzed by information provided by the Ministry of Culture and supplemented by field work in Taiwan. Informational accessibility concentrates on digital accessibility. With the establishment of the Ministry of Digital Affairs in 2021, Taiwan seems to have acknowledged the importance of digital transformation. Web access is an important tool for obtaining information in the digital age, but it can be difficult for people with disabilities to navigate, especially those with visual impairments. Web Content Accessibility Guidelines (WCAG) serve as an international standard for the accessibility of web content. The WCAG is a standard implemented throughout all government websites, as well as national museum websites in Taiwan. Emotional accessibility is depicted by case studies in various national museums because how museums choose to create an emotional connection to its visitors with disabilities is not standardized
Ritual Performance in the American Feminist Utopian and Dystopian Novel from the Twentieth to the Twenty-First Century
Full text linkThis thesis explores the significance of ritual acts in feminist speculative fiction as a lens through which to examine the construction of gender, power, and resistance in both utopian and dystopian contexts. Against the backdrop of socio-political developments since the beginning of the twenty-first century - marked by increasing attacks on women’s rights and growing global inequality - the resurgence of feminist dystopian narratives reflects a growing cultural unease about stalled progress toward gender equality. This context provides a compelling rationale for examining the renewed popularity of feminist speculative fiction in recent decades and the narrative functions of ritual therein.
The analysis focuses on a selection of feminist speculative novels from the North American literary tradition, written over the span of more than a century. These texts represent a wide spectrum of feminist visions and fall into three broad categories: utopian, dystopian, and hybrid forms. A shared cultural background among the authors allows for a consistent comparative analysis grounded in ritual theory as developed in Western academic contexts.
Drawing on theoretical concepts from ritual studies - particularly those related to performance, social order, and symbolic meaning - the thesis argues that rituals in these works function not only as mechanisms of control used by oppressive regimes but also as transformative and symbolic acts of resistance. Rituals surrounding themes such as gender roles, reproductive rights, family structures, and political participation are shown to play a central role in maintaining or subverting existing power relations. In dystopian narratives, ritual practices often reinforce hierarchical, patriarchal systems, while in utopian visions they can enable solidarity, agency, and the construction of alternative social frameworks.
The methodological approach combines literary analysis with insights from gender theory and anthropology. This interdisciplinary framework highlights how feminist speculative fiction uses ritual as a structural and thematic device to critique dominant ideologies and to imagine new forms of community and identity
Mixed Views on Community Building in Mixed-use Spaces : An Investigation on Mixed-use Spaces and its potential to increase Trust and Community Building among Residents or increase Social Mixes in Toronto, Canada
Full text linkMixed-use spaces, which is having various land uses near home, are increasing in several neighborhoods of Toronto as a solution to some issues, such as affordability and increasing a sense of community. The data about the residents’ experience in a formal and informal mixed-use space identifies the residents’ community building, and it provides an initial understanding if mixed-use spaces are a viable planning option for the future. To analyze the concept of community building and mixed-use spaces, a lens of the urban dimension of trust is beneficial, as it focuses on the relational experiences that residents have in the spaces, through the act of interacting by trusting other residents, which can lead to community building. Planners, architects, developers, and neighborhood associations impact the ways in which the relations in these spaces form. To fully understand the dynamics, a trust-related concept, which is authority, is also necessary to note the power relations that can influence the residents’ community building in their neighborhoods. This study reveals the importance of planning city spaces based on the residents’ needs since they live in these areas, and it provides new insights on the links between the urban dimension of trust, mixed-use neighborhoods, and community building
The Phase Diagram of QCD at High Densities
Full text linkIn this thesis, we investigate the phase structure of Quantum Chromodynamics (QCD) at high densities, with the aim of locating the critical endpoint (CEP) of its temperature crossover transition. To this end, we utilise the functional Renormalization Group (fRG) to directly compute non-perturbative QCD from first principles. Aiming for quantitative predictions, we both develop important numerical tools and investigate the systematics of functional QCD to correctly assess the systematic error of our approach.
Hydrodynamic methods are introduced as a powerful tool to resolve full field dependences in general fRG setups. We develop a local Discontinuous Galerkin (LDG) Finite Element (FE) method for unprecedented quantitative precision and reliability for this task. For the task of resolving corresponding effective potentials, we develop new classes of regulators for dynamical spontaneous symmetry breaking (dSSB) and find optimal RG-time integration algorithms for the highly stiff partial differential equations (PDEs) of such systems.
The vertex expansion employed for the glue sector of functional QCD requires a careful treatment of tensor bases, and we discuss optimisation aspects thereof in detail. Furthermore, we introduce the LEGO® principle to formalise the modular structure of functional QCD, which we will use for our systematic error estimates.
We show the quantitative reliability of our approach in vacuum QCD and illustrate that the finite temperature limit thereof is in great agreement with results from other first-principle approaches.
For the first time in the fRG, a reliable systematic error estimate is provided for the corresponding observables, in particular for the pseudo-critical temperature Tpc = 156.7 ± 0.6 MeV. We resolve the QCD phase structure up to baryon chemical potential µB = 1000 MeV and find a sizeable regime with a potential inhomogeneous instability covering the potential critical endpoint (CEP) of QCD
Investigation of a remote 32-channel whole-body coil for large field of view 1H body imaging at 7 T
Full text linkThis work aimed to evaluate the remote coil in comparison to existing local coils and to experimentally investigate the parallel transmit (pTx) capabilities of the remote coil through in-vivo measurements. The thesis was divided into two parts.
First, the transmit and receive performance of seven different local pTx coils and the remote coil (also in combination with a dedicated local receive array) were quantitatively assessed through phantom measurements. The remote coil demonstrated the largest excitation coverage in head-foot direction. Although, its maximum B1+ efficiency at the isocenter was 2.0 times lower than that of local transmit-receive coils, this limitation could be compensated with the four- or eight-fold higher total available power. While the best performing local transmit-receive coil enabled higher acceleration factors than the dedicated local receive array, the latter achieved the highest signal-to-noise ratio in the phantom’s center.
Second, the pTx capabilities of the remote coil were investigated through in-vivo measurements. The first study demonstrated homogeneous liver excitation using static pTx.
The second in-vivo study extended the excitation region to the whole abdomen, where two kT-point pTx pulses were required for sufficient homogeneity across all subjects.
In conclusion, the remote coil, combined with a dedicated local receive array, represents the state-of-the-art for body imaging at 7 T. These findings highlight its potential for large field-of-excitation imaging and its necessity for translating UHF body MRI into clinical practice
The study on myeloid-derived suppressor cells (MDSCs) and T cells in melanoma patients with adverse events after immunotherapy immune checkpoint inhibitors
Full text linkImmune checkpoint inhibitors (ICI) represent a remarkable breakthrough in melanoma treatment. The therapy could considerably improve treatment outcomes, particularly for advanced melanoma patients. However, despite the great success of ICI therapy, approximately 30-60% of patients experience immunological side effects, known as immune-related adverse events (irAE). IrAE can be triggered by an overactivation of the immune system following ICI treatment. These adverse events can sometimes be fatal, leading to lasting organ damage, the requirement for systemic immunomodulatory treatment, and even treatment discontinuation. Therefore, biomarkers capable of predicting irAE onset or identifying patients at risk of experiencing irAE are crucial for effective diagnosis and management.
IrAE development might be attributed to an imbalanced immune system, which can be mediated by increased T cell activity or a loss of function of immunosuppressive cell subsets like regulator T cell (Tregs).
In the present study, I aimed to establish an immune profile in the peripheral blood of melanoma patients associated with irAE onset. For this purpose, I conducted routine laboratory tests and flow cytometry analysis of peripheral blood samples from 31 melanoma patients treated with anti-PD-1 monotherapy or anti-PD-1-/anti-CTLA-4 combination therapy. I investigated the activation status of T cells and the role of immunosuppressive subsets like Tregs and monocytic myeloid-derived suppressor cells (M-MDSCs) in patients with and without irAE. My analysis included different time points: before ICI start, during ICI treatment, at the onset of irAE, and during immunosuppressive treatment to manage irAE.
Overall, I observed a significantly improved progression-free survival (PFS) among patients with irAE. Additionally, I demonstrated an activation of CD8+ T cells indicated by an upregulation of the early activation marker CD69, and an increased frequency of activated CD4+ T cells (CD4+CD25+FOXP3-) during irAE. Furthermore, I revealed a decrease in Tregs during irAE occurrence. Moreover, lower frequencies of Tregs correlated with more severe adverse events.
Another aim of this study was to evaluate the impact of immunomodulatory drugs following irAE on circulating immune cell subsets. Here, I observed that the number of M-MDSCs and Tregs tended to be elevated during immunosuppressive treatment.
Analysis of routine blood laboratory tests found increased LDH and CRP serum levels during adverse events.
Taken together, the present study identified that certain activated T cell subsets and the decrease of Tregs may lead to an imbalanced immune homeostasis, which could potentially promote the occurrence of irAE