Strukturelle und funktionelle Untersuchungen am Cytochrom-bc1-Komplex zur Klärung des molekularen Wirkungsmechanismus

The cytochrome bc1 complex is a cornerstone in bioenergetic electron transfer chains, where it carries out tasks as diverse as respiration, photosynthesis, and nitrogen fixation. This homodimeric multisubunit membrane protein has been studied extensively for several decades and the enzyme mechanism is described with the modified protonmotive Q cycle. Still, the molecular and kinetic description of the catalytic cycle is not complete and questions remain regarding the bifurcation of electron transfer at the quinol oxidation (Qo) site, substrate occupancy, pathways of proton conduction, and the nature of the Rieske protein domain movement. We used competitive inhibitors to study the molecular architecture at the Qo site with X-ray crystallography. The structure of the enzyme with the substrate analog 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole (HHDBT) bound at the Qo site was determined at 2.5 Å resolution. Spectroscopic studies showed that HHDBT is negatively charged when bound at the active site. Mechanistic interpretations from inhibitor binding are in line with single occupancy model for quinol oxidation and structural analysis supports the proposed proton transfer pathway. For functional insight into the enzyme mechanism, redox-sensitive protonation changes were studied by Fourier transform infrared spectroscopy. The protein purification procedure was optimized for less delipidation and the isolated enzyme was more active. Furthermore, two new phospholipids were identified in the X-ray structures, including a cardiolipin. Strikingly, conserved lipid binding cavities were observed in structural comparison with homologous enzymes. The functional role of tightly bound phospholipids will be discussed. Finally, the Qo site is a target for various compounds of agricultural and pharmaceutical importance. Importantly, the X-ray structures permit detailed analysis of the molecular reasons for acquired resistance to and treatment failure of Qo site inhibitors, such as atovaquone, that is used to treat malaria and pneumonia, as discussed herein.Der Cytochrom-bc1-Komplex ist eine essentielle Komponente von Elektronen-transportketten in Eukaryoten und Prokaryoten, wo er an verschiedenen bioenergetischen Prozessen wie der Atmungskette, der Photosynthese und der Stickstoffixierung beteiligt ist. Der Mechanismus dieses Enzyms ist in seinen Grundzügen charakterisiert und mit dem Model des modifizierten Q-Zyklus beschrieben. Jedoch sind Regulationsprozesse und insbesondere der molekulare Mechanismus der Ubichinoloxidation immer noch unverstanden. Um den molekularen Mechanismus der Katalyse in der Qo-Bindungsstelle zu untersuchen, wurde der Bindungsmodus des Qo-Inhibitors HHDBT mittels der Röntgenkristallographie bestimmt. Diese 2.5 Å Kristallstruktur erlaubte wichtige Rückschlüsse auf den Katalysemechanismus. Vorherigen kristallographischen Arbeiten am UHDBT-Inhibitorkomplex waren nicht erfolgreich und führten nicht zu einer hochaufgelösten Struktur. Unter Verwendung von Heptyl-HDBT, das eine kürzere Alkyl-Kettenlänge aufweist, wurden erfolgreich dreidimensionale Kristalle des Komplexes gezüchtet. Dieses Hydroxychinon bindet in seiner ionisierten Form, entgegen vorherigen Behauptungen, dass eine ionisierte Verbindung nicht in der katalytischen Substrattasche binden könne. Demnach weisen unsere Studien darauf hin, dass die pH-Abhängigkeit der alkyl-HDBT Inhibierung durch eine dissoziierbare Gruppe im Komplex, vermutlich den Liganden des [Fe-2S]-Zentrums His181, bewirkt wird und nicht durch eine Verringerung der Inhibitoreffizienz durch Deprotonierung der Hydroxylgruppe. Die strukturelle Ähnlichkeit zu Ubichinol und die kompetitive Hemmung der Qo-Bindungsstelle erlauben es, HHDBT als Substratanalogon zu betrachten. Die Konformationsänderungen an der Bindungsstelle unterstützen den zuvor postulierten Protonentransferweg und offenbaren die Plastizität der katalytischen Bindungsstelle. Aus dem beobachteten Bindungsmodus des Hydroxychinon-Anions und dem Protonentransferweg wurde ein katalytischer Mechanismus abgeleitet, der sich mit dem Modell einer einfachen Besetzung der Ubichinol-Oxidationstasche in Einklang bringen lässt. Um das vorgeschlagene mechanistische Modell zu überprüfen, wurde die FTIR- Spektroskopie eingesetzt. Diese Studie zeigte zum ersten Mal, dass die Bindung der Inhibitoren HHDBT und Stigmatellin den Protonierungszustand und/oder die Orientierung von sauren Seitenketten beeinflusst. Des weiteren wurde die Interaktion der Stigmatellin Carbonylgruppe mit dem Enzym nachgewiesen. Die Röntgenkristallstrukturen der Hefe Cytochrom-bc1-Komplexe mit gebundenen HHDBT oder Stigmatellin in der Qo-Bindungsstelle sind wertvoll um die molekularen Ursachen menschlicher Krankheiten zu verstehen, die auf Mutationen im Cytochrom b zurückzuführen sind. Ebenfalls von großer Bedeutung sind diese Strukturen für die gezielte Entwicklung von Pestiziden oder pharmazeutischen Wirkstoffen, die gegen das aktive Zentrum der Komplexe in Parasiten oder Pilzen gerichtet sind. Zielorganismen entwickeln oft eine Resistenz gegenüber diesen Wirkstoffen. Bei einem Vergleich von Cytochrom-bc1-Komplex Strukturen von Hefe und Rind, mit verschiedenem Besetzungsgrad der Chinon-Bindungsstellen wurde die Plastizität der Qo-Bindungsstelle beobachtet. Basierend auf dem HHDBT Bindungsmodus in der Qo-Bindungsstelle wurde die Interaktion mit z.b. dem Antimalariawirkstoff Atovaquone modelliert. Die strukturellen Grundlagen von dessen Spezies-spezifischer Wirkung können so diskutiert werden. Ein weiteres Ergebnis der Doktorarbeit war die Optimierung der Proteinreinigung im Hinblick auf eine geringere Delipidierung der Proteinprobe und eine vergrößerte Kapazität. Dabei wurde die Ausbeute verbessert sowie die spezifische Enzymaktivität des gereinigten Proteins erhöht. So konnten zwei neue Phospholipidmoleküle reproduzierbar in unterschiedlichen Datensätzen identifiziert werden. Eine Analyse des Bindungsmodus von fest gebundenen Lipiden in Membranproteinstrukturen wurde durchgeführt. Es wurden hierbei spezifische Bindungsmuster für die Stabilisierung der Interaktionen zwischen den Phosphodiestergruppen und den Seitenketten der Aminosäurereste identifiziert. Eine bevorzugte Stabilisierung der Phospholipide auf der negativen Seite der Membranen wurde beobachtet. Die in der Kristallstruktur identifizierten, spezifisch gebundenen Phospholipide, die an Interaktionsflächen von Proteinuntereinheiten auftreten, weisen auf eine wichtige Rolle für die strukturelle Integrität des Komplexes hin. Eine vergleichende Analyse der Lipidbindungsstellen in Strukturen homologer Proteine wurde durchgeführt und zeigte, dass sie Spezies-übergreifend konserviert sind. Die Röntgenstrukturen des Cytochrom-bc1-Komplexes bieten die Grundlage für detaillierte Struktur-Funktions-Studien zur Beschreibung der molekularen katalytischen Vorgänge. Sie fördern aufregende Entdeckungen für das tiefere Verständnis der fundamentalen Prozesse des Lebens, welche durch diesen Typ der Enzyme katalysiert werden

Risk prediction in prostate cancer diagnostics : current challenges and improvements

Prostate cancer has been considered a disease of elderly men, and thus historically less focus has been on prostate cancer research than many other cancer types. However, as life expectancy is increasing all over the world, more life years are lost when men are diagnosed with prostate cancer at the age of 70 years now than before. Therefore, it is increasingly important to improve the diagnostic pathway of prostate cancer in modern health care. My thesis aims to address some of the issues in the current prostate cancer diagnostic pipeline using risk prediction mod-els. Measuring the level of prostate-specific antigen (PSA) in blood is widely used as a blood test to screen for prostate cancer and evidence has shown that mortality decreases with PSA testing. However, because PSA testing has a high false-positive rate, many unnecessary biopsies are per-formed on healthy men and many men are overdiagnosed with indolent disease (International Society of Urological Pathology (ISUP) grade group 1). In Study I the objective was to predict the risk of clinically consequential cancer (ISUP ≥ 2) at biopsy and the cumulative probability of having a negative biopsy when being PSA tested with one, two, three, four, or five to eight year intervals. We found that men with a PSA level above 1 ng/mL had an increased risk of ISUP ≥ 2 prostate cancer when screened with longer then annual intervals, while men with a PSA level below 1 ng/mL had low risk of ISUP ≥ 2 prostate cancer regardless of time between testing. The benefit of a shorter screening interval needs to be balanced with the increased cumulative probability of having a negative biopsy which we found to be twofold for annual vs. biennial testing intervals and threefold for annual vs. triennial testing intervals. Knowledge about the relationship between PSA, age and different grades of prostate cancer is important for clinicians working with prostate cancer diagnosis because of how widely used the PSA test is. In Study II we studied the association between the risk of indolent and clinically consequential prostate cancer (ISUP 1 and ISUP ≥ 2) and PSA and age, respectively. Our study cohort comprised of 6.083 biopsied men from the STHLM3 study and 72.996 biopsy cores from those men. In the overall ISUP grade system, lower grades can be masked by higher grades, and thus we studied the associations for both overall ISUP grade and for ISUP grade on each biopsy core. Our results showed that ISUP 1 prostate cancer was not significantly associated with PSA or age, on overall ISUP grade or on individual biopsy core level. In contrast, our results showed that ISUP ≥ 2 prostate cancer is significantly associated with increasing PSA level and older age. Our results indicate that PSA leakage of ISUP 1 prostate cancer cells is more similar to that of benign prostate tissue than ISUP ≥ 2 prostate cancer tissue. The use of magnetic resonance imaging (MRI) before biopsy to diagnose prostate cancer has increased in current clinical practice. Combining results from prostate MRI with existing risk prediction models can improve the predictive abilities of the models. The aim of Study III was to develop a risk prediction model (S3M-MRI), combining the Stockholm3 score and the PI-RADS (Prostate Imaging Reporting and data System) score from MRI to predict the risk of ISUP ≥ 2 prostate cancer. We developed the S3M-MRI model using data from the STHLM3-MRI diagnostic study and compared the model performance of the S3M-MRI to the Stockholm3 model and PI-RADS score. We also compared five diagnostic strategies for clinical outcomes. We found that the combined S3M-MRI model had better predictive abilities than both the Stockholm3 and the PI-RADS alone. However, when we compared it to different clinical strate-gies, the sequential use of the Stockholm3 test followed by MRI on Stockholm3 positive men resulted in similar numbers of performed biopsies and diagnosed ISUP ≥ 2 prostate cancers while saving many MRI scans. Prostate cancer diagnosis is based on the result of the prostate biopsy and reclassification of ISUP grade on radical prostatectomy samples compared to biopsy is common. In Study IV our aim was to study what effect reclassification of disease status based on prostate biopsies has on the performance of prostate cancer risk prediction models using simulations and data from the STHLM3 Radical Prostatectomy Cohort. The cohort comprised of 780 men from the STHLM3 study who were diagnosed with prostate cancer and treated with a radical prostatec-tomy between 2013 and 2015. We compared four simulated prediction model scenarios with and without error in disease status and calculated the area under the receiver operating charac-teristics (ROC) curve (AUC) of the Stockholm3 score for predicting clinically significant prostate cancer assessed using biopsy and radical prostatectomy samples. Our simulations showed that fitting a risk prediction model using data with error in the disease status only leads to a small decline in the true predictive performance, but leads to a large decline in apparent predictive performance when evaluated against data with error in the disease status. Moreover, our results showed that the Stockholm3 test has stronger association with clinically significant prostate cancer defined on prostatectomy samples (without errors) than on biopsy samples (with errors). In conclusion, in this thesis we have aimed to describe a part of the risk associated with diag-nosis of prostate cancer as well as developing new prostate cancer risk prediction models. This thesis contributes to the constant pursue of improving the current prostate cancer diagnostic pipeline in order to improve the lives of men screened for or diagnosed with prostate cancer

Phase field damage simulations of debonding between matrix and spherical inclusions

Polymer-bonded explosives (PBX) are complicated composites, and it is important for practical applications to know how they deform and fracture. PBX consists of large volume fraction of energetic particles and experiments show that the interface is a critical region for structural stability since particles are often weakly bonded to the polymer and cracks initiate there and the composite fractures. This study focuses on the interface between a particle and a binder and how the particle inclusion starts to debond from the polymer binder in compressive and tensile loading. A phase field damage model is used to model the response of a glass bead particle in a Sylgard 184 binder under compressive loading, and the model is validated with a Kolsky bar experiment of the same set-up. The comparison between the simulations and the experiment reveals that the damage model should be described with positive volumetric strain only, and deviatoric strain does not contribute to damage in compressive loading of Sylgard 184. In tensile loading, the phase field damage model is used to study cavitation and debonding, as compared with results from literature. In cavitation the interface is perfectly bonded and the fracture initiates slightly away from the interface. In debonding the interface is weaker than the matrix, and fracture initiates at the interface. Analytic solution of the critical stress needed for debonding shows good agreement with the simulations from the model

Does More Time in a Therapeutic Garden Lead to a Faster Return to Work? A Prospective Cohort Study of Nature-Based Therapy, Exploring the Relationship between Dose and Response in the Rehabilitation of Long-Term Patients Suffering from Stress

Background: Stress-related mental illness is increasing worldwide and leading to long-term illness. Most of those affected are aged 30-50, so the need for rehabilitation and return to work for these patients is great. Research indicates that staying in nature can lead to stress recovery. The question is whether nature-based therapy can rehabilitate people who suffer from long-term stress-related mental illness, and how long a period of rehabilitation is necessary. Methods and findings: The research was carried out at Alnarp Rehabilitation Garden, which is a specially designed health garden on the Swedish University of Agricultural Sciences’ campus area, where the participants in the study were treated by a licensed rehabilitation team. The intention was to examine three cohorts of participants prospectively. These were offered different lengths of a nature-based rehabilitation program through a natural experiment. Participants were referred to Alnarp Rehabilitation Garden from three local social insurance agencies that granted different lengths of rehabilitation programs: 8 weeks, 12 weeks and 24 weeks. The length of the program was determined by which local social insurance agencies they belonged to, not the participants' level of illness. Primary outcome was return to work. Other outcomes were occupational function, personal control and sense of coherence. The results showed that all three rehabilitation interventions gave significantly good results, but that longer nature-based rehabilitation led to significantly better results for all outcomes. The 12-week program provided 75% greater return to paid work and the 24-week program 120% greater return to paid work than the 8-week program. Conclusion: There is a significant positive relationship between treatment time in the rehabilitation garden and return to work. The study also indicates that the effects may level off after twelve weeks. More studies are needed to further investigate the relationships

3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.

Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features

Forest Therapy for Women with Gynaecological Cancer—A Feasibility Study to Find New Alternatives in Cancer Rehabilitation

Cancer can have a significant impact on one’s life situation, with many patients reporting psychosocial discomfort, worry, anxiety, fear of recurrence, depression, tiredness/fatigue, sleep problems, pain and numerous other problems even long after active medical treatment. Psychosocial support during cancer rehabilitation has proven to be insufficient. In a recent debate article, the scientific committee of CancerRehabFund, Sweden, demands more rehabilitation alternatives for individuals living with cancer. Nature-based treatment is one of the alternatives mentioned as the way forward, but more research is needed. Therefore, we want to evaluate the patient’s experience of a ten-week forest bathing intervention, as an add-on to the standard care, and whether it can improve general health and well-being in women suffering or recovering from gynaecological cancer. The study will run between the autumn of 2022 and until the end of 2023. It is a prospective single-case study, including quantitative and qualitative approaches using validated self-administered instruments (pre–post measurements) and semi-structured interviews (post) on women’s lived experience of the 10-week forest bathing intervention. The quantitative outcome measurements will be the quality of life, fatigue and depression/anxiety. There will also be a questionnaire on perceived sensory dimensions experienced in the forest environment. The study will include 24 participants, divided into four groups of 6 participants. Once a week for ten weeks, the participants will be offered a session of a 2.5-hour stay in the forest with breathing exercises, slow movement, time in silence and privacy and a social gathering to conclude each session. Before and after each session, the participants will be invited to fill in the Profile of their mood state to describe their mood/feelings. There will be three different forest locations with varied forest cover types, i.e., evergreen, deciduous and mistands. Participation in this study will be voluntary, and all results will be anonymously presented on a group level. This paper is a protocol paper describing in detail the venues/forest sites, the forest therapy intervention and the scientific methodological approach for evaluating the ten-week intervention. To our knowledge, this is the first study on forest bathing for cancer survivors in Sweden. The Swedish Ethical Review Authority has approved the study [Dnr 2022-02083-01]

Health and lifestyle : Icelanders' everyday life information behaviour

Diss. Åbo Akademi UnversityIncludes bibliographical references (p. 216-237)