Quantitative Imaging of Protein-Protein Interactions by Multiphoton Fluorescence Lifetime Imaging Microscopy using a Streak camera

Fluorescence Lifetime Imaging Microscopy (FLIM) using multiphoton excitation techniques is now finding an important place in quantitative imaging of protein-protein interactions and intracellular physiology. We review here the recent developments in multiphoton FLIM methods and also present a description of a novel multiphoton FLIM system using a streak camera that was developed in our laboratory. We provide an example of a typical application of the system in which we measure the fluorescence resonance energy transfer between a donor/acceptor pair of fluorescent proteins within a cellular specimen.Comment: Overview of FLIM techniques, StreakFLIM instrument, FRET application

Development of a Multiphoton Fluorescence Lifetime Imaging Microscopy (FLIM) system using a Streak Camera

We report the development and detailed calibration of a multiphoton fluorescence lifetime imaging system (FLIM) using a streak camera. The present system is versatile with high spatial (0.2 micron) and temporal (50 psec) resolution and allows rapid data acquisition and reliable and reproducible lifetime determinations. The system was calibrated with standard fluorescent dyes and the lifetime values obtained were in very good agreement with values reported in literature for these dyes. We also demonstrate the applicability of the system to FLIM studies in cellular specimens including stained pollen grains and fibroblast cells expressing green fluorescent protein. The lifetime values obtained matched well with those reported earlier by other groups for these same specimens. Potential applications of the present system include the measurement of intracellular physiology and Fluorescence Resonance Energy Transfer (FRET) imaging which are discussed in the context of live cell imaging

Democracy in the People of God? Investigations in the Acts of the Apostles

Unter der Fragestellung nach dem Umgang mit Macht in der jungen Kirche werden relevante Stellen aus der Apostelgeschichte (Apg) analysiert: die Übertragung von Macht durch die Wahl der Sieben (Apg 6,1-7); die ermaechtigende Beauftragung von Barnabas und Saulus (Apg 13,1-3); die gemeinsame Wahrnehmung von Macht durch die Jerusalemer Versammlung (Apg 15,1-41); schliesslich das Vermaechtnis des Paulus in der Miletrede (Apg 20,17-38). Synchrone Methoden (angelehnt an die narrativen Analysemethoden von G. Genette und S. Rimmon-Kenan) und die klassische historische Kritik ergaenzen sich dabei. Seitenblicke zeigen die Analogien zwischen dem Leben der christlichen Gemeinden und anderen Organisationen (bspw. Vereine, juedische Diasporasynagoge) in ihrem zeitgenoessischen Umfeld, die Lukas besonders betont. Mit Hilfe der Macht-Theorie von H. Arendt werden die Ergebnisse aus der Apg als Zeugnis für das Bewusstsein einer gemeinsamen Macht aller im Volk Gottes interpretiert, die in demokratischen Formen ihren Ausdruck fand. Auf der Basis der grundlegenden Christenrechte - Freiheit, Gleichheit und Geschwisterlichkeit -, wie sie das NT bezeugt, werden schliesslich Grundzüge einer demokratischen Reform der katholischen Kirche erarbeitet.Under the question, how the early christian church dealt with power, relevant passages from the Acts of the Apostles (Acts) are exegetically analysed in this thesis: the transfer of power within the election of the Hellenistic Seven (Act 6:1-7); the empowering commission of Barnabas and Saul by the Antiochian christian community (Act 13:1-3); the common practising of power in the Jerusalem assembly (Act 15:1-41); finally the last will of Paul, given in his farewell address at Milet (Act 20:17-38). In doing so, synchronous methods (following the narrative analysing-methods of G. Genette and S. Rimmon-Kenan) and the classic instruments of the historical criticism complete one another. Some Sideglances are able to point out the analogies - especially emphasized by Luke - between the life of the early christian communities and other organisations in their contemporary environment (e.g. associations or the Jewish synagogue). Following the theory of power, given by H. Arendt, the results from the analysis of Acts are interpreted as a testimony for the consciousness of the common power of all members in the people of God, which is expressed in democratical forms of organizing. Based upon the fundamental rights of all christians - freedom, equality an brotherhood -, as the New Testament proofs them, basics for a democratic reform of the catholic church are finally presented

Die Heilkraft der menschlichen Hand : ein Beitrag zur Lehre und richtigen Anwendung der Heilkräfte des Lebens-Magnetismus

von Julius Neubert

SPDEF Regulates Fatty Acid Metabolism Driving Tumor Growth in Androgen Receptor-Positive Breast Cancer

Despite advances in the treatment of breast cancer, it is still the second leading cause of cancer-related death in women worldwide. A large number of patients develop recurrence and die of advanced metastatic disease. More than 70% of metastatic breast cancers express androgen receptor (AR) representing a potential target for anti-hormone therapies. AR is suggested to directly interact with the lineage-specific transcription factor, SAM pointed domain-containing ETS transcription factor (SPDEF) in breast cancer however, the functional role of SPDEF and its interaction with AR remains to be elucidated. I found that AR expression highly correlates with SPDEF expression in breast cancer patients. To study its functional role in tumorigenesis and metastatic outgrowth, I utilized patient-derived xenograft (PDX) derived cell lines from liquid biopsies of metastatic breast cancer patients. Using genetically manipulated PDX cell models, I demonstrated that repression of SPDEF significantly reduced tumor growth of AR+ breast cancer cells in vivo. Downregulation of SPDEF prevented metastasis formation in the brain whereas lung metastatic lesions were not affected by SPDEF silencing. Reduced tumor growth upon downregulation of SPDEF was also observed in estrogen receptor (ER)-positive breast cancer cells. Notably, I observed enhanced tumor growth in an AR negative breast cancer model suggesting a tumor suppressive function when AR is not present. Overexpression of SPDEF in AR- breast cancer cells significantly inhibited in vivo tumor growth. To investigate the underlying mechanism on the molecular level, I established transcriptional profiles by performing tumor tissue and cell line microarray analysis in SPDEF-overexpressing and knockdown models. Mechanistically, I found that SPDEF regulates key metabolic processes: (1) Pharmacological inhibition of AR or silencing of SPDEF restricted mitochondrial respiration activity resulting in decreased energy production. AR activation by testosterone treatment enhanced basal and maximal oxygen consumption rates, as did SPDEF overexpression. However, testosterone treatment did not restore decrease in mitochondrial respiration when SPDEF was downregulated. (2) Further, I found that SPDEF regulates genes encoding enzymes involved in glucose and fatty acid metabolism in AR+ breast cancer cells. FBP1, the rate-limiting enzyme in gluconeogenesis was identified as a direct target gene of SPDEF. However, FBP1deletion did not impair in vivo tumor growth. Enzymes involved in de novo fatty acid biosynthesis were downregulated in SPDEF knockdown SPDEF-deficient cells. The fatty acid transporter CD36 was upregulated upon downregulation of SPDEF as validated by RT-qPCR and western blot analysis. Flow cytometry analysis revealed increased plasma-membrane localized CD36 expression in shSPDEF cells and vive versa, cell surface CD36 expression was decreased in SPDEF-overexpressing cells. I performed isotope tracing experiments using 13C-glucose, 13C-glutamine and 13C-acetate to functionally assess fatty acid metabolism upon deregulation of SPDEF. SPDEF knockdown cells showed decreased biosynthesis of specific fatty acids, however, they restored their cellular fatty acid pool by increased uptake of exogenous fatty acids. Abolishing CD36 expression in AR+ breast cancer cells suggested that fatty acid uptake is critically required for cell growth of SPDEF knockdown cells. Pharmacological inhibition of CD36 induced a significant cytostatic effect in SPDEF knockdown cells. These data suggest that CD36 mediates exogenous fatty acid uptake as compensatory pathway when de novo fatty acid biosynthesis is decreased by SPDEF downregulation. In agreement, SPDEF knockdown cells did not have a significant growth disadvantage in vitro under saturated culture conditions. Usual cell culture media contain saturated levels of carbon sources and nutrients which do not reflect the physiological conditions found in the patient, making it difficult to study the metabolic profiles of cancer cells in vitro. However, when cells were cultured under physiological conditions resembling the natural cellular environment found in the patient, cells showed a significantly decreased growth rate when SPDEF was downregulated. These findings suggest that lipid and energy metabolism are transcriptionally regulated by SPDEF facilitating cell survival in nutrient-depleted environments and hence, tumor and metastatic outgrowth of AR+ breast cancer cells. Since initial data suggested that pharmacological inhibition of AR mimics the effect of SPDEF downregulation, targeting AR and CD36 simultaneously may be a treatment strategy for AR+ breast cancer patients