STED Nanoscopy to Illuminate New Avenues in Cancer Research – From Live Cell Staining and Direct Imaging to Decisive Preclinical Insights for Diagnosis and Therapy

Molecular imaging is established as an indispensable tool in various areas of cancer research, ranging from basic cancer biology and preclinical research to clinical trials and medical practice. In particular, the field of fluorescence imaging has experienced exceptional progress during the last three decades with the development of various in vivo technologies. Within this field, fluorescence microscopy is primarily of experimental use since it is especially qualified for addressing the fundamental questions of molecular oncology. As stimulated emission depletion (STED) nanoscopy combines the highest spatial and temporal resolutions with live specimen compatibility, it is best-suited for real-time investigations of the differences in the molecular machineries of malignant and normal cells to eventually translate the acquired knowledge into increased diagnostic and therapeutic efficacy. This thesis presents the application of STED nanoscopy to two acute topics in cancer research of direct or indirect clinical interest. The first project has investigated the structure of telomeres, the ends of the linear eukaryotic chromosomes, in intact human cells at the nanoscale. To protect genome integrity, a telomere can mask the chromosome end by folding back and sequestering its single-stranded 3’-overhang in an upstream part of the double-stranded DNA repeat region. The formed t-loop structure has so far only been visualized by electron microscopy and fluorescence nanoscopy with cross-linked mammalian telomeric DNA after disruption of cell nuclei and spreading. For the first time, this work demonstrates the existence of t-loops within their endogenous nuclear environment in intact human cells. The identification of further telomere conformations has laid the groundwork for distinguishing cancerous cells that use different telomere maintenance mechanisms based on their individual telomere populations by a combined STED nanoscopy and deep learning approach. The population difference was essentially attributed to the promyelocytic leukemia (PML) protein that significantly perturbs the organization of a subpopulation of telomeres towards an open conformation in cancer cells that employ a telomerase-independent, alternative telomere lengthening mechanism. Elucidating the nanoscale topology of telomeres and associated proteins within the nucleus has provided new insight into telomere structure-function relationships relevant for understanding the deregulation of telomere maintenance in cancer cells. After understanding the molecular foundations, this newly gained knowledge can be exploited to develop novel or refined diagnostic and treatment strategies. The second project has characterized the intracellular distribution of recently developed prostate cancer tracers. These novel prostate-specific membrane antigen (PSMA) inhibitors have revolutionized the treatment regimen of prostate cancer by enabling targeted imaging and therapy approaches. However, the exact internalization mechanism and the subcellular fate of these tracers have remained elusive. By combining STED nanoscopy with a newly developed non-standard live cell staining protocol, this work confirmed cell surface clustering of the targeted membrane antigen upon PSMA inhibitor binding, subsequent clathrin-dependent endocytosis and endosomal trafficking of the antigen-inhibitor complex. PSMA inhibitors accumulate in prostate cancer cells at clinically relevant time points, but strikingly and in contrast to the targeted antigen itself, they eventually distribute homogenously in the cytosol. This project has revealed the subcellular fate of PSMA/PSMA inhibitor complexes for the first time and provides crucial knowledge for the future application of these tracers including the development of new strategies in the field of prostate cancer diagnostics and therapeutics. Relying on the photostability and biocompatibility of the applied fluorophores, the performance of live cell STED nanoscopy in the field of cancer research is boosted by the development of improved fluorophores. The third project in this thesis introduces a biocompatible, small molecule near-infrared dye suitable for live cell STED imaging. By the application of a halogen dance rearrangement, a dihalogenated fluorinatable pyridinyl rhodamine could be synthesized at high yield. The option of subsequent radiolabeling combined with excellent optical properties and a non-toxic profile renders this dye an appropriate candidate for medical and bioimaging applications. Providing an intrinsic and highly specific mitochondrial targeting ability, the radiolabeled analogue is suggested as a vehicle for multimodal (positron emission tomography and optical imaging) medical imaging of mitochondria for cancer diagnosis and therapeutic approaches in patients and biopsy tissue. The absence of cytotoxicity is not only a crucial prerequisite for clinically used fluorophores. To guarantee the generation of meaningful data mirroring biological reality, the absence of cytotoxicity is likewise a decisive property of dyes applied in live cell STED nanoscopy. The fourth project in this thesis proposes a universal approach for cytotoxicity testing based on characterizing the influence of the compound of interest on the proliferation behavior of human cell lines using digital holographic cytometry. By applying this approach to recently developed live cell STED compatible dyes, pronounced cytotoxic effects could be excluded. Looking more closely, some of the tested dyes slightly altered cell proliferation, so this project provides guidance on the right choice of dye for the least invasive live cell STED experiments. Ultimately, live cell STED data should be exploited to extract as much biological information as possible. However, some information might be partially hidden by image degradation due the dynamics of living samples and the deliberate choice of rather conservative imaging parameters in order to preserve sample viability. The fifth project in this thesis presents a novel image restoration method in a Bayesian framework that simultaneously performs deconvolution, denoising as well as super-resolution, to restore images suffering from noise with mixed Poisson-Gaussian statistics. Established deconvolution or denoising methods that consider only one type of noise generally do not perform well on images degraded significantly by mixed noise. The newly introduced method was validated with live cell STED telomere data proving that the method can compete with state-of-the-art approaches. Taken together, this thesis demonstrates the value of an integrated approach for STED nanoscopy imaging studies. A coordinated workflow including sample preparation, image acquisition and data analysis provided a reliable platform for deriving meaningful conclusions for current questions in the field of cancer research. Moreover, this thesis emphasizes the strength of iteratively adapting the individual components in the operational chain and it particularly points towards those components that, if further improved, optimize the significance of the final results rendering live cell STED nanoscopy even more powerful

Betriebswirtschaftliche Analyse von ökologisch wirtschaftenden Großbetrieben in Ostdeutschland

Die vorliegende Arbeit hat das Ziel, die Wirtschaftlichkeit von ökologisch wirtschaftenden Grossbetrieben in Ostdeutschland zu untersuchen. Dazu ist es erforderlich, die sozioökonomische Situation von ökologisch wirtschaftenden Großbetrieben auf der einen und die Vermarktungsbedingungen auf der anderen Seite zu erfassen. Im Rahmen dieser Studie wurde deshalb über zwei Erhebungspfade a) die Befragung von 43 ökologischen Großbetrieben und b) Gespräche mit 30 Experten im Vermarktungssektor in Ostdeutschland eine breite empirische Datenbasis erstellt. Die Studie zeigt, dass bei der strategischen Ausrichtung der Betriebe wirtschaftliche Motive in den Vordergrund rücken. So wurde insbesondere bei Betrieben in der Rechtsform juristischer Personen in großem Maße Arbeitskräfte abgebaut bei gleichzeitiger Erweiterung der Flächenkapazitäten. Des weiteren hat die Bedeutung von Investitionen in die Vermarktung der eigenen Produkte deutlich an Bedeutung zugenommen. Darüber hinaus erfolgte eine zunehmende Spezialisierung bei Mutterkuhbetrieben. Ein wesentlicher Nachteil von Grossbetrieben sind die höheren Transaktionskosten Exemplarisch wurde für Milchviehhaltende und Mutterkuhhaltende Großbetriebe die Wettbewerbsfähigkeit gesamtbetrieblich untersucht. Dazu wurden auf der Basis der empirischen Erhebungen vier Betriebsmodelle definiert, die sich hinsichtlich ihrer Faktorausstattung, Rechtsform und Betriebsform unterscheiden. Für die untersuchten Milchviehbetriebe zeigt sich, dass die ökologische Milchproduktion auf Großbetrieben zur ähnlichen bis besseren Betriebsergebnissen führen kann als eine konventionelle Milchproduktion. Bei Mutterkuhhaltenden Großbetrieben ist der Betriebserfolg in hohem Maßen an staatlichen Transferleistungen (Mutterkuhprämie, Schlachtprämien, Extensivierungsprämien) gekoppelt. Nur bei dem Betriebsmodell, das die Mutterkuhhaltung auf Betrieben in der Rechtsform natürlicher Personen repräsentiert konnte ein positives Betriebsergebnis erzielt werden

The combinatorics of interval-vector polytopes

An \emph{interval vector} is a $(0,1)$-vector in $\mathbb{R}^n$ for which all the 1's appear consecutively, and an \emph{interval-vector polytope} is the convex hull of a set of interval vectors in $\mathbb{R}^n$. We study three particular classes of interval vector polytopes which exhibit interesting geometric-combinatorial structures; e.g., one class has volumes equal to the Catalan numbers, whereas another class has face numbers given by the Pascal 3-triangle.Comment: 10 pages, 3 figure

Note From the Editor

Background and AimsTo review published studies on the effectiveness of combining cognitive-behavioural therapy (CBT) and motivational interviewing (MI) to treat comorbid clinical and subclinical alcohol use disorder (AUD) and major depression (MDD) and estimate the effect of this compared with usual care. MethodsWe conducted systematic literature searches in PubMed, PsycINFO and Embase up to June 2013 and identified additional studies through cross-references in included studies and systematic reviews. Twelve studies comprising 1721 patients met our inclusion criteria. The studies had sufficient statistical power to detect small effect sizes. ResultsCBT/MI proved effective for treating subclinical and clinical AUD and MDD compared with controls, with small overall effect sizes at post-treatment [g=0.17, confidence interval (CI)=0.07-0.28, Pless than0.001 for decrease of alcohol consumption and g=0.27, CI: 0.13-0.41, Pless than0.001 for decrease of symptoms of depression, respectively]. Subgroup analyses revealed no significant differences for both AUD and MDD. However, digital interventions showed a higher effect size for depression than face-to-face interventions (g=0.73 and g=0.23, respectively, P=0.030). ConclusionsCombined cognitive-behavioural therapy and motivational interviewing for clinical or subclinical depressive and alcohol use disorders has a small but clinically significant effect in treatment outcomes compared with treatment as usual

Re-thinking Housing and Mobility – A European Living Lab for Sustainable Mobility in Munich

This paper aims to describe the vision and implementation approach of a sustainable and innovative mobility and housing concept of a city district at the pericentral edge of Munich. Within the European CIVITAS initiative, the ECCENTRIC project demonstrates an innovative approach to mobilize residents by offering intermodal mobility and mobility on demand. With around 8000 new inhabitants and 12,000 new employees within the next years, the transport system in the Munich living lab Domagkpark and Parkstadt Schwabing needs an integrative and innovative approach to ensure a functioning, ecologically compatible and socially acceptable mobility supply. Central objective is to increase quality of life in the district through a substantial roll-out of innovative mobility solutions, that reduce the use (and number) of private cars.With the implementation of various project measures in the field of sustainable and shared mobility, mobility management, city logistics and road security, a new model quarter for sustainable urban development and compatible mobility will be development. Successful research findings aim to be implemented in future newly-built quarters of Munich and replicated in other European cities

Resource partitioning between the oligolectic leafcutter bee Megachile lapponica (Hymenoptera, Apiformes) and other visitors on flowers of Epilobium angustifolium (Onagraceae)

Das schmalblättrige Weideröschen, Epilobium angustifolium, zählt in Mitteleuropa zu den charakteristischen Pionierpflanzen auf Kahlschlagflächen und Waldrändern. Durch seine lange Blütezeit (Juni bis September) ist es für viele Blütenbesucher eine wichtige Pollen- und Nektarressource im ansonsten blütenarmen Spätsommer (MAURITZIO & SCHÄFER 1994). Zu seinen häufigsten Blütenbesuchern zählt die solitär lebende oligolektische Blattschneiderbiene Megachile lapponica. Sie sammelt Pollen nur an Pflanzen der Gattung Epilobium und bevorzugt dabei das schmalblättrige Weidenröschen (WESTRICH 1989). Aber auch Honigbienen, Apis mellifera, sind oft in großer Menge an diesen Blüten anzutreffen. Sie nutzen E. angustifolium vorwiegend als Nektarquelle (MAURITZIO & SCHÄFER 1994), sammeln aber auch Pollen auf den Blüten. Bei starkem Beflug durch die Honigbienen könnte es daher zu einer Verknappung der Ressource Pollen kommen. Dies könnte dazu führen, dass die Wildbienenweibchen für ihre Sammelflüge mehr Zeit und Energie aufwenden müssen. Im kritischen Fall einer Konkurrenz sollte auch die Aufzuchtrate und somit die Fitness von M. lapponica betroffen sein, deren Larven fast ausschließlich mit Epilobium-Pollen verpflegt werden. Auf einer Kahlschlagfläche im Kottenforst (Bonn) sollte untersucht werden, welche Insekten an den Blüten des schmalblättrigen Weidenröschens Pollen und/oder Nektar sammeln und welche Blütenbesucher gleichzeitig auch Blütenbestäuber sind. Vor allem aber sollte die Frage geklärt werden, ob es durch die Honigbiene zur Konkurrenz um den Pollen kommt.In a clear-felling in the Kottenforst (Bonn) we registered the abundance of flower visiting insects on Epilobium angustifolium and observed their behaviour during flower visits. The most frequent bees on the flowers were Megachile lapponica, an oligolectic bee on Epilobium, Apis mellifera L. and Bombus spec. Furthermore syrphid flies and beetles were observed. Recordings of the seed set after single visits to flowers showed that syrphid flies and bumblebees were more efficient pollinators than M. lapponica and honeybees. In order to study the potential competition between honeybees and M. lapponica, we increased the density of honey bees in two trials of 14 days each by bringing 15 bee hives with approximately 15.000 individuals per colony to the study site. Before and during the presence of additional honeybees we recorded the frequencies of visits by M. lapponica on pilobium-flowers, measured the duration of foraging flights and the numbers of brood cells constructed per day by single M. lapponica females. In the pollen traps of the bee hives we recorded the number of pollengrains and the relative amount of Epilobium-pollen collected by honeybees. We found that honey bees collected 440 g pollen per day. The portion of Epilobium-pollen in the pollen traps of the honey bees was very low with mean 0,97 % (SD=4,89, n=251). During the increased density of honeybees we recorded neither a decline in the frequency of flower visits by M. lapponica, nor an increase in duration of foraging trips. Furthermore, no decline in the number of brood cells built and provisioned by M. lapponica could be registered. Thus, our results of this case study do not indicate a negative influence of the increased abundance of honey bees on the fitness of M. lapponica

Identification of possible cow grazing behaviour indicators for restricted grass availability in a pasture-based spring calving dairy system

peer-reviewedPrecision livestock farming uses biosensors to measure different parameters of individual animals to support farmers in the decision making process. Although sensor development is advanced, there is still little implementation of sensor-based solutions on commercial farms. Especially on pasture-based dairy systems, the grazing management of cows is largely not supported by technology. A key factor in pasture-based milk production is the correct grass allocation to maximize the grass utilization per cow, while optimizing cow performance. Currently, grass allocation is mostly based on subjective eye measurements or calculations per herd. The aim of this study was to identify possible indicators of insufficient or sufficient grass allocation in the cow grazing behaviour measures. A total number of 30 cows were allocated a restricted pasture allowance of 60% of their intake capacity. Their behavioural characteristics were compared to those of 10 cows (control group) with pasture allowance of 100% of their intake capacity. Grazing behaviour and activity of cows were measured using the RumiWatchSystem for a complete experimental period of 10 weeks. The results demonstrated that the parameter of bite frequency was significantly different between the restricted and the control groups. There were also consistent differences observed between the groups for rumination time per day, rumination chews per bolus and frequency of cows standing or lying