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    RNA Interference as a Vector Control Mechanism: Reducing Aedes albopictus Populations and Disrupting Arbovirus Transmission Cycles
    (2025) Omokungbe, Bodunrin
    Mosquitoes are considered the “most dangerous animals on Earth”. This is not because of the small amount of blood they take from us, but due to the pathogens they can transmit during this process. Key examples are malaria parasites, dengue virus, chikungunya virus, and Zika virus, causing over a million deaths annually. Urbanization, transport, and global trade have led to the spread of invasive species such as the Asian tiger mosquito (Aedes albopictus). Originally from Southeast Asia, this species has migrated to other parts of the world. This mosquito can transmit numerous arboviruses, filarial worms, and bacteria. Conventional control relies on chemical insecticides and biological agents, but off-target effects and resistances limit their usefulness. Therefore, targeted approaches like RNA interference (RNAi) are essential. RNAi is a naturally occurring post-transcriptional gene silencing mechanism in most eukaryotes. Silencing essential genes via RNAi can induce mortality, distort vital phenotypes, and impair the ability to transmit pathogens. This thesis evaluated RNAi as a species-specific control strategy against Ae. albopictus. Prior successes have demonstrated improving RNAi outcome in other mosquito species using transfection reagents (TRs), so I hypothesized that formulating dsRNA with TRs would enhance uptake and efficacy. However, no TRs are specifically designed for long dsRNA in aedine cell lines, and their undisclosed compositions makes selection difficult. Here, I established an RNAi workflow for aedine cell lines and screened multiple TRs for dsRNA delivery. Their complexing capacity and the cytotoxicity of their complexes were assessed. Most of them formed stable complexes, except HiPerFect, which failed even at a 1:9 (dsRNA:TR) ratio. The complexes were mostly non-cytotoxic, but Lipofectamine 2000 exhibited cytotoxic effects at concentrations above 1 ng/μL. Meanwhile, the five most effective TRs increased cellular uptake of long dsRNA and improved RNAi knockdown efficiency in Ae. albopictus U4.4 cells. Furthermore, candidate genes associated with high mortality in other insects were selected and two dsRNA constructs per gene were designed. Initial evaluation in U4.4 cells was conducted with both unformulated and TR-encapsulated dsRNA. Only one dsRNA against inhibitor of apoptosis (IAP) reduced U4.4 cell viability, yet all selected dsRNA showed significant knockdown of the candidate genes by RT-qPCR. Subsequently, I established RNAi workflow for the in vivo assessment in Ae. albopictus, but no dsRNA led to significant larval mortality. The knockdown of IAP gene was observed, but only in dissected gut tissue, and not in the whole body. The lack of larval mortality led to further investigations to identify possible barriers limiting RNAi efficacy. Particle sizing indicated optimal dsRNA:TR complex sizes, but only at lower concentrations. Fluorescence imaging confirmed oral uptake, but no spread of the dsRNA beyond the gut. Ex vivo assays showed rapid dsRNA degradation by larval gut extract, which were identified in Ae. albopictus for the first time and are expressed across larval stages, with the highest expression in gut tissues. The data indicated that the lack of larval mortality was likely due to suboptimal particle size (at higher concentrations), poor systemic spread, and rapid degradation of the selected dsRNA by nucleases. In addition, a standardized protocol was developed to analyze alphavirus replication in aedine cell lines. Viral inhibition was demonstrated with furin inhibitors using a SFV reporter tagged with mCherry as a model. This workflow thereby provides an in vitro platform for evaluating dsRNA against mosquito-borne viruses. Lastly, the feasibility of RNAi to reduce SFV replication in aedine cell line was assessed using the established protocol. For this, dsRNAs were designed and showed no cytotoxicity. Most of the synthesized dsRNAs inhibited virus spread when encapsulated. The dsRNA against non-structural protein 4 (nsp4) reduced viral replication by up to 80%. A concentration of 0.5 ng/μL of the encapsulated dsRNA was enough to significantly suppress the spread of the reporter virus signal. The antiviral effect of nsp4-dsRNA was validated by RT-qPCR, which confirmed a significant knockdown of the target gene. The central hypothesis that encapsulation of dsRNA increases efficacy was supported by most of the cell line experiments. However, the in vitro successes did not translate to in vivo lethality. Therefore, future work should develop optimized formulations to protect dsRNA and promote spread beyond the larval gut. More so, identifying gut-essential genes could enable larval mortality without systemic spread. While suppression of arboviral replication in an aedine cell line was demonstrated here, in vivo validation is still required. A potential RNAi bioinsecticide or arboviral transmission inhibitor must be potent, economical, and highly target-specific. Overall, this thesis presented the first comprehensive analysis of TRs for aedine cells, developed an RNAi workflow for evaluating dsRNA in Ae. albopictus, established a protocol to measure alphavirus infection in real time, and also showed that RNAi can reduce arboviral replication in mosquito cells.
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    Zahl der metastabilen Zustände bei einem Neuronalen Netzwerk mit Projektorkopplungen
    (1990) Kuhlmann, Peter
    Der Mittelwert der Zahl der metastabilen Zustände eines Neuronalen Netzwerkes mit Projektorkopplungen wird analytisch berechnet. Der Mittelwert wird dabei über zufällig verteilte Muster gebildet. Die Berechnung erfolgt mit Hilfe der Sattelpunktmethode.
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    Advances in Site-Selective Acylation of Pyranosides: From Oligopeptide Catalysis to Immobilized Catalysts in Flow
    (2025) Seitz, Alexander
    In the search for potential new applications for our group’s catalysts, we explored the idea of combining two catalytic motifs, that we have extensively studied before, in a multicatalytic reaction. The initial step involves performing a site-selective acetylation of partially protected pyranosides using a π-methyl-histidine (PMH)-containing oligopeptide catalyst. The resulting mono-alcohols could then be utilized in a selective, thiourea-catalyzed glycosylation reaction to generate 2-deoxy disaccharides. This thesis focuses on the first reaction of the proposed sequence. In the first part, we present the site-selective acylation of various methyl 4,6-O-protected pyranosides. We screened several tetrapeptide catalysts containing PMH and an adamantane moiety as the backbone. We identified catalysts capable of overcoming the intrinsic reactivity, which we determined using N-methylimidazole (NMI), for most of the pyranosides studied. To optimize the reaction conditions, we employed design of experiments (DOE) studies. We also investigated the impact of the 4,6-O-protecting group and the acylation reagent. Furthermore, we demonstrated that the selectivity of the reaction increased with the length of the applied peptide catalyst, suggesting that hydrogen-bonding interactions play a crucial role in selectivity. Finally, we showed that the observed reactivity could be maintained in more complex systems, as we successfully combined a benzylidene protection and the site-selective acetylation in a one-pot reaction. In the second part, we investigated the same pyranosides using peptide catalysts immobilized onto Wang-resin. We developed this approach to enhance the sustainability of our reaction, but also after initial studies concerning multicatalysis with thiourea and oligopeptide catalysts showed interference between the two, indicating that they must be separated locally. During the study, we demonstrated that we could easily synthesize the immobilized catalysts via solid-phase peptide synthesis (SPPS) and that they were still able to overcome the intrinsic reactivity of the substrates. We found that the catalysts could be reused for several reaction cycles with consistent results. Additionally, we showed that we could apply the catalyst in a continuous flow reaction without a significant loss in reactivity and selectivity. We used the long-term activity of the catalyst to convert large quantities of substrate and observed that the catalyst’s selectivity remained intact even after a temporary change in substrate.
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    Deciphering the role of EZH2 in the control of HIF2α signaling and its effects on cellular phenotypes in breast and lung cancer
    (2026) Kruijning, Salisa
    Hypoxia is a hallmark of solid tumors and a critical driver of cancer progression, largely mediated by hypoxia-inducible factors (HIFs). While HIF1α has been extensively studied, the transcriptional regulation of HIF2α, encoded by the endothelial PAS domain protein 1 (EPAS1) gene, remains less well understood. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), is a known epigenetic regulator with canonical roles in gene repression via H3K27 tri-methylation and emerging non-canonical functions, including transcriptional activation. The interplay between EZH2 and HIF signaling, particularly regarding HIF2α, is largely unexplored. In this study, the role of EZH2 as a potential regulator of HIF2α in breast and lung cancer model systems was investigated. EZH2 knockdown decreased HIF2α protein under hypoxic conditions and EPAS1 mRNA levels under normoxic and hypoxic conditions in MDA-MB-231 and PC-9 cell lines. Restoration of EZH2 rescued EPAS1 expression. Mechanistic studies revealed that this regulation occurs independently of PRC2, EZH2’s methyltransferase activity, EZH1, Notch1 signaling, and transcriptional elongation. Chromatin immunoprecipitation demonstrated direct binding of EZH2 to a region approximately 1.7 kb downstream of the EPAS1 transcription start site, without enrichment of H3K27me3, supporting a non-canonical transcriptional activator function for EZH2. These findings indicate that EZH2 directly maintains EPAS1 transcription independent of chromatin repression and contributes to sustaining transcriptional activity within a globally repressive hypoxic environment. Functionally, EZH2 depletion impaired the expression of HIF2α target genes, including GLUT1 and PGK1, and reduced invasion capacity in MDA-MB-231 cells under hypoxia. In PC-9 cells, EZH2 knockdown decreased proliferation, which was partially rescued by transient HIF2α restoration, and anchorage-independent growth. These findings indicate that EZH2 promotes tumorigenicity at least in part through HIF2α. Clinical analyses revealed that high EZH2 and EPAS1 expression correlate with poor prognosis in breast cancer patients, underscoring the potential clinical relevance of this regulatory axis. Overall, this study establishes EZH2 as a novel non-canonical transcriptional activator of EPAS1, linking an epigenetic regulator to hypoxia signaling. These findings extend our understanding of EZH2 beyond its canonical repressive role and suggest that the EZH2-HIF2α regulatory axis may contribute to malignant phenotypes such as proliferation and invasion under hypoxic conditions. Future studies utilizing RNA-seq, in vivo models, and pharmacological inhibitors targeting EZH2 and HIF2α could provide further insight into the mechanistic and therapeutic potential of this regulatory pathway in cancer.
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    Lokales Wissen und kommunale Anpassung an den Klimwandel. Geographische und politologische Analysen für eine verbesserte Umsetzung
    (2026) Gierhake, Klaus
    The Context: This work exists as an update to the study: ‘Municipal adaptation to climate change in Quito (Ecuador) from the perspective of governmentality’ (http://dx.doi.org/10.22029/jlupub-5762). Here, two major content areas are analysed in more detail: 1) The local knowledge of a metropolitan area. 2) Social innovation as a process of combining different parts of existing local knowledge. This study focuses on institutions, their political and administrative actions, and areas with intersecting functions. Methodologically, this research is based on the field of applied geography. Therefore, a step-by-step refinement of this research approach is used. The Climate Convention: International conventions on global issues are highly complex. This applies to the Climate Convention as well. Here the focus is on discussions and resolutions relating to adaptation, the implementation of technical solutions in local communities. At COP27 in Egypt (2023), the following points were made exceptionally clear: (1) Progress in adaptation is very limited, varies greatly from region to region, and is highly fragmented. Adaptation measures could, in principle, have a number of additional positive effects. (2) There are a number of barriers, in particular the fact that the vast majority of international climate change funding has been concentrated on mitigation measures. Only a small proportion has been allocated to adaptation so far. This has been financed primarily from public funds. (3) Positive starting conditions can be identified, e.g. clear adaptation goals, responsibilities, and commitments to support. In particular, improved local knowledge of the risks, impacts and possible options is crucial. New forms of governance are possible. Local Knowledge: The discussion about local knowledge in large metropolitan areas is very recent. Metropolitan areas are the national centres of universities. They have a large civil society, and are often very diverse, bringing together immigrants from all parts of the country with their different cultures and forms of knowledge. This initial structure of great social diversity represents enormous potential. Local knowledge is then the sum of different segments of knowledge, from all groups and institutions, in very different sub-areas, and in one and the same space. Social innovations (‘Oslo Declaration’): This approach is complex in terms of content, combining ecological, social and economic objectives. However, the concept also addresses an even more comprehensive view: social innovations could also arise from combining different components of locally existing knowledge, focused on a new issue, e.g. adaptation to climate change. This necessitates a certain re-evaluation of general scientific discourse. Even scientific content, such as publications, that may appear ‘outdated’ at first glance, due to a sectoral perspective or simply because of the publication date, gains value in a new context. In this case, this can be demonstrated for the modernisation of a metropolis and adaptation to climate change. This requires implementation of qualitative research approaches, at least in the initial phase. Introduction of an innovation and territorial Diffusion: However, almost no examples from the Global South have been published on this topic. When this has occurred, it has been for projects with a very narrow understanding of ‘social’ (health, education) and a very limited project scope. On the other hand, there are examples of how local knowledge outside universities has been successfully structured and used to introduce social innovation. Such innovations can clearly also be introduced in the Global South. There is little empirical work available, although the example of Quito shows a completely new trend in geographical innovation research. Diffusion is possible even without widespread dissemination in the immediate local context. Existing institutional networks have enabled a ‘leap’ to the international level. Significant progress could be achieved by taking adequate account of local knowledge and its importance in social innovation approaches, including in international strategies. Some examples include: (1) The “UN Millennium Development Goals”, here in particular with reference to sustainable cities. (2) The German Research and Innovation Agenda ‘City of the Future’, especially with regard to the installation of social innovation in research and implementation. (3) The resolution of the ‘UN World Summit on Sustainable Development’ on a new perspective on ‘corporate social responsibility’ as an instrument for monitoring public policy. (4) Local adaptation to climate change as a starting point, and the results of the programme “100 Resilient Cities”. Outlook: The example of ‘Ecuador - Quito’ shows that considerable conceptual progress has been made. At the same time, the comprehensive level of ‘social innovation in Quito’ documents a remarkable global diffusion process of scientific results. This is highly significant in that this approach explicitly includes the ecological aspect of innovation in its concept and, in the specific case of Quito, also contains a remarkable document on sector planning. The local strategy for adapting to climate change deserves attention. The successful diffusion process of information therefore incorporates this technical aspect (climate change) without any additional effort. On this basis, regional governance strategies can be developed that offer a new quality of location factors. For example, a centre for identifying local (urban knowledge) and its possible combinations in different territorial contexts. Application-oriented/multidisciplinary and medium-term research remains necessary in order to overcome current knowledge deficits. Central research perspectives are identified: - Local knowledge, on the part of universities and the respective civil society. - The intellectual capital of local government, as an essential source of local knowledge can be used in a targeted manner. - The reasons for the emergence/existence of barriers are fundamentally complex. This necessarily includes the perspective of the effects of such barriers on a diffusion process. - A stronger territorial-institutional perspective is necessary in such research. The role of metropolitan areas should be examined more specifically in terms of their perspectives as centres of local knowledge and comprehensive social innovation, - In parallel with the completion of the current case study, it is necessary to undertake socio-political investigations into the role of individual actors, their values and attitudes towards social, ecological and economic changes.