Page 51/60 Annual Report GCB 2025In the third results chapter of this work, we present a straightforward purification strategy for mammalian NADH:ubiquinone oxidoreductase (complex I), cytochrome bc1 complex (complex III) and ATP synthase (complex V). Starting from isolated bovine mitochondria, the sequential purification strategy allows to isolate bovine respiratory complexes for structural and functional studies. First, we present the 3.0 Å structure of the entire bovine cytochrome bc1 complex stabilized with the peptide-scaffold peptidisc. While the structure of bovine complex III has been solved and studied multiple times, the process of solving the here presented model might be of methodological relevance for future work. This model was obtained with cryo-EM and subsequent in silico purification, an approach that allows to solve multiple structures of macromolecules with the same grid. This strategy is useful for semi-pure protein mixtures that need to be structurally analyzed on individual level, thereby reducing the need for a highly pure sample obtained with wet lab techniques. In the second part of this project, we provide experimental evidence for the successful co-reconstitution of various respiratory complexes of mammalian origin. The simplest system achieved here is based on a proton pump, the cytochrome c oxidase, and the ATP synthase, both co-reconstituted together in artificial vesicles entrapped with cytochrome c. This setup allows to investigate oxidative phosphorylation inhibitors without the risk of potential cross-reactivities of inhibitors with other biological targets in cellular environment. Doseresponse experiments using this minimal respiratory-driven system revealed a more pronounced inhibitory effect of oligomycin A over venturicidin A, both macrolides being important and well-characterized ATP synthase inhibitors.This minimal setup for cellular respiration was sequentially expanded by incorporating bovine cytochrome bc1 complex and later also NADH:ubiquinone oxidoreductase. While we concede that this system has to be further optimized (e.g. stoichiometry of respiratory complexes, ATP synthesis quantification), to our knowledge, the successful interplay of four mammalian respiratory complexes in an artificial membrane demonstrates the first synthetic respiratory chain (without complex II) enabling ATP production from NADH via electron-mediators coenzyme Q10 and cytochrome c. This experimental setup was further used to investigate the impact of the mitochondrial signature phospholipid cardiolipin on artificial ATP production, in which experiment we observed slightly increased ATP synthesis in liposomes containing cardiolipin.In the main part of this PhD thesis, we investigated the mode of action of leucinostatin A and its derivative lefleuganan (ZHAWOC6027) on mitochondrial ATP synthase and mitochondrial functionality in general. Leucinostatin A is a natural antimicrobial peptide that exhibits strong protozoal but also mammalian toxicity. In contrast, lefleuganan is a simplified fully synthetic leucinostatin A derivative that shows a substantially decreased systemic toxicity in a rat model. Both compounds share their activity against various protozoan pathogens with IC50 values in the low nanomolar range in cell culture experiments, thus rendering lefleuganan a promising antiprotozoal drug candidate. To date, lefleuganan is in phase I clinical trial for treatment of cutaneous leishmaniasis.In the present work, we demonstrate that leucinostatin A acts as a specific ATP synthase inhibitor of different organisms including mammals (H. sapiens, B. taurus), while this inhibitoryeffect is lost in lefleuganan. This is a fundamental discovery and an indispensable prerequisite for lefleuganan to become a clinically relevant compound.Using an established ATP synthase inhibitor competition assay, we identified the leucinostatin A binding site within the ATP synthase that includes the proton binding motif Glu59 present in the membrane-embedded subunits c of the ATP synthase. Having two structurally similar compounds in our hands, one of which is an ATP synthase inhibitor and the other not, we set out to identify the responsible moieties that mediate specific ATP synthase inhibition. In a comprehensive structure-activity relationship with a myriad of leucinostatin A derivatives, we identified the hydroxyleucine at position 7 in leucinostatin A, replaced with a simple leucine in lefleuganan, as the determinant for ATP synthase inhibition. While the complete structure activity relationship was conducted with a liposome-based ATP-producing system consisting of bo3 oxidase and bovine ATP synthase, a selection of compounds was tested on human fibroblast cells using high-resolution respirometry. Convincingly, in agreement with the previous results, all peptides having the hydroxyleucine at position 7 showed ATP synthesis inhibition, while all others with a canonical leucine at this position did not inhibit ATP synthase. The combination of those experiments unanimously confirms position 7 of leucinostatin A and its derivatives to act as a molecular switch thereby potentially mediating ATP synthase inhibition, depending on the particular moiety present at this position. We further observed that leucinostatin A and lefleuganan act as membrane potential dissipating agents, with lefleuganan surpassing leucinostatin A in this particular effect.Our results further suggest that antiprotozoal activity of leucinostatin A (derivatives) is not mediated by protozoan ATP synthase inhibition. Here, based on experiments with small and giant lipid vesicles, we present experimental evidence that leucinostatin A derivatives damage the integrity of biological membranes in a detergent-like manner, a process that is stimulated in the presence of negatively charged lipids. We attribute the activity of those peptides on membrane integrity and permeability to be the causative property for hypertoxicity towards protozoan species containing a single mitochondrion (e.g. Leishmania spp).Taken together, we identified the responsible moiety of leucinostatin A conferring ATP synthase inhibition in a profound structure-activity relationship study. In addition, we report that antiprotozoal activities of
Page 52/60 Annual Report GCB 2025leucinostatin A and its derivatives are not mediated by ATP synthase inhibition, but are caused by efficient modulation of membrane integrity and permeability. The here provided data support the start of clinical studies with lefleuganan.Finally, a high-resolution structure of the ATP synthase in complex with leucinostatin A is required to unravel the complete interaction network between drug and target. Therefore, cryo-EM is currently being established as a novel methodology in our group. A brief description of this process and preliminary results are given in an excursus chapter.
Page 53/60 Annual Report GCB 20255.3.2 Student Awards 20255.3.3 Stem Cell Research and Regenerative Medicine Platform (SCRM) 2nd Annual Award Stem Cell Research and Regenerative Medicine Platform (SCRM) first annual award The Stem Cell Research and Regenerative Medicine Platform (SCRM) announced in 2023, a new prize for the most outstanding PhD thesis in the field of stem cell and/or regenerative medicine research. Supervisors were invited to nominate their students until September 2024 for this award. Following the nominations, a shortlist of three students were selected to deliver brief presentations at the SCRM Annual Meeting in November 2025. The winner of the award was determined during the annual meeting, and the award was presented to Dr. Paola Bermudez Lekerika for her thesis, “Interpreting intervertebral disc phenotypes: An in-vitro, ex-vivo and in-silico approach.”Low back pain (LBP) is a chronic and highly prevalent condition, which not only is considered a leading cause of disability worldwide, but also affects people from all ages. Although LBP aetiology is multifactorial, intervertebral disc (IVD) degeneration is considered a key contributor, accounting for ~40% of chronic LBP cases. An IVD consists of an avascular proteoglycan-rich nucleus pulposus (NP) laterally constrained by the peripheral fibre-reinforced annulus fibrosus (AF), situated between two cartilage endplates (CEP) of adjacent vertebral bodies. Under physiological conditions, the extracellular matrix (ECM) metabolism is balanced between anabolic and catabolic processes. However, during degeneration different structural and biochemical alterations occur which imbalance ECM metabolism and the action of anabolic and catabolic mediators, contributing to the loss of proteoglycans and decreased disc hydration, which may lead to blood vessel ingrowth, innervation and pain. Hence, depending on the degenerative degree, different IVD phenotypes can arise with diverse ECM metabolic and mediator components. Thus, this thesis aims to interpret different IVD phenotypes according to the degree of degeneration, culture system and environmental condition utilizing an in-vitro, ex-vivo and in-silico approach. As a result, this dissertation has contributed to the previous knowledge i) by a novel understanding of sulfated alginate hydrogels for NP cell 3D culture, ii) by developing a stimulation model for IL-1β and TNF factors defining dose and time in bovine NP and AF cells, iii) by elucidating a NP cell trauma or degenerated phenotype-dependent role of multiple mediators including IL-4, IL-10, IL-13 and finally by iv) building and evaluating in-silico NP-specific protein-protein network models to interpret and prioritize key protein candidates under different IVD phenotypic scenarios and IL-4, IL-10 and IL-1β conditions. Altogether, i) sulfated hydrogels are not recommended for NP cell culture, ii) standardized IVD culture systems defining environmental conditions (i.e. oxygen tension, culture system, media components) are needed to further evaluate IL-1β and TNF titration effects, iii) the role of IL4 and IL-1β might be determined by different IVD phenotypes, while IL-10 may be linked to angiogenic processes iv) PPI networks could elevate experimental data analysis and propose novel protein candidates.
Page 54/60 Annual Report GCB 20256. Facts and Figures6.1 Highlights | 2025Registrations and Graduations | 2025
Page 55/60 Annual Report GCB 20256.2 Highlights | Graduates2025Graduations by Faculty | 2025 Graduates6.2.1 Graduations by Expert Committee | 2025 Graduates
Page 56/60 Annual Report GCB 20256.2.2 Degree Titles by Faculty | 2025 Graduates6.2.3 Country of Origin | 2025 Graduates
Page 57/60 Annual Report GCB 20256.3 Five-Year Figures (2021-2025)6.3.1 Graduates’ Gender Distribution6.3.2 Median Years to Graduate
Page 58/60 Annual Report GCB 20257. Digital Presence7.1 Communication and Social MediaFollow GCBLinkedInJoined in June 2023. Over 968 followers by end of 2025.GCB WebsiteThesis defenses, graduations, publications and more featured here.GCB101 ILIASGCB101: Everything students and supervisors need to know about how to navigate the GCB processes and requirements in clear, easy-to-understand steps.30% increase, from over 800 members end of year 2024 to 1040 members by end of year 2025.
Page 59/60 Annual Report GCB 20258. AcknowledgmentsWe thank the University of Bern Leadership and Deans of the GCB Faculties of Medicine, Science, and Vetsuisse for their support in administering the graduate school. This support enables us to maintain excellence in our structured graduate program.From the Academic Careers Office we would like to thank Vice Rector Prof. Andrew Chan and Marco Hollenstein for their efforts to champion support for the next generation PhD students at the GCB.We thank the GCB PhD Committee for their support in maintaining the program's quality amidst a high volume of new applications. We appreciate their efforts in overcoming the challenges of running a competitive and productive graduate school of this scale.GCB Mentors: Thank you for your dedication to our PhD students, valuable input on research proposals, and support during challenging times. Your guidance is highly appreciated and often praised by graduates.Raffaele Battaglia and the Institute of Social and Preventive Medicine (ISPM) IT Support team (Ives Gerber and Christian Wyniger): Thank you for your unflappable support throughout varying levels of IT literacy and many urgent requests. You have kept us in business and prevented serious work stoppages due to IT challenges.PhD Specialization program coordinators and staff: Your innovation in improving the GCB curriculum by providing students the opportunity to specialize their study programs further, opening more career avenues, and elevating the GCB to a first-rate graduate school is commendable.The CTS/KSL team (Norbert Wernicke, Roger Hasler, Jan Müller, Marina Beutler): Your expertise, quick and knowledgeable responses to our questions and issues, and quick wit in mitigating system-related frustration are greatly appreciated.Thank you to Irina Polisi for the support in HR-related questions.Thank you to Barbara Schär and the team in the finance department for answering our questions and continuing to provide your support.
Page 60/60 Annual Report GCB 2025University of BernGraduate School for Cellular and Biomedical Sciences (GCB)Mittelstrasse 433012 BernTelephone Email Website+41 31 684 59 61 [email protected] https://www.gcb.unibe.ch/