Multiplexed biochemical imaging reveals the extent and complexity of non-genetic heterogeneity in DNA damage-induced caspase dynamics.

Genetically identical cells show a heterogeneous response to a multitude of signals such as growth factors and DNA damage. While this heterogeneity has been shown to be a major determinant of treatment success in several diseases including cancer, little is known about how differences in biochemical signalling networks underlie such heterogeneity. State-of-the-art methodologies to study biochemical networks are often invasive and enable to quantify biochemical events only on cell populations or at a single point in time for a single cell, and therefore, cannot adequately quantify the fast, asynchronous and heterogeneous responses. In order to address these limitations, we have developed a unique sensing platform based on fluorescence lifetime imaging microscopy (FLIM) capable to multiplex at least three biosensors by utilizing Förster Resonance Energy Transfer (FRET) efficiently. After an overall introduction in Chapter 1, I describe the rational design and characterization of novel FRET pairs aiming to utilize the visible spectrum efficiently in combination with FLIM in Chapter 2. We combined blue, green and red donor fluorescent proteins that are excited at the same wavelength (840 nm for two-photon excitation) with genetically encoded quenchers, i.e. non-fluorescent chromoproteins as acceptors. This sensing platform enables the simultaneous detection of three biochemical reactions within single living cells providing new opportunities to characterize and understand non-genetic heterogeneity. In Chapter 3, I will demonstrate the first application of this novel platform by studying the activity of three key enzymes in DNA damage-induced cell death, caspase-2, -3, and -9. We confirm the heterogeneous nature of Cisplatin-induced cell death in genetically identical cells but reveal the existence of at least three subpopulations of cells characterized by distinct caspase dynamics. By combining biochemical and morphological information we infer the existence of different biochemical network topologies that are associated with alternative death phenotypes each cell adopts, such as apoptosis and programmed necrosis. Finally, deconvolution of cellular populations and direct measurement of a three-node caspase network - formerly impossible - permitted us to design perturbations of cell fate choices utilizing clinically relevant inhibitors. These perturbations resulted in changes in cell fate in response to Cisplatin, a clinically desirable outcome that suggests new avenues for combinatorial drugging and a new strategy to reveal cancer vulnerabilities that may be otherwise confounded by typical genetic and non-genetic heterogeneity.Gates Cambridge Trus

Herzklopfen im Anamnesegespräch: eine Erfassung von Stress in unterschiedlichen Gesprächssituationen

In der medizinischen Ausbildung werden täglich Simulationen zum Erlernen von kommunikativen und praktischen Fähigkeiten eingesetzt und sind nicht mehr wegzudenken. Übungen mit unterschiedlichen Realitätsgraden und verschiedenen Gesprächspartner/-innen (Kommiliton/-innen, Schauspielpatient/ innen, echte Patient/-innen) werden zum Erlernen von Anamnesegesprächen eingesetzt, ohne in diesem Kontext etwas über den Stress von Medizinstudierenden zu wissen. Ziel der Studie war es, das Stresslevel von Medizinstudierenden während des Anamnesegesprächs bei Gesprächsszenarien unterschiedlichen Realitätsgrades mit psychischen und physischen Stressparametern zu erfassen und zu vergleichen. Hierfür wurden Medizinstudierende der Universität Tübingen im 2. Studienjahr (N = 128, Rücklaufquote: 76.6%) im Wintersemester 2018/19 zu der Studie eingeladen und zufällig auf eines von drei Gesprächsszenarien zur Übung der Anamnese verteilt. In den Gesprächsszenarien wurde entweder ein Anamnesegespräch mit Kommiliton/ innen (RS), mit Schauspielpatient/-innen (SP) oder mit echten Patient/-innen (EP) geführt. Die psychische Stresskomponente der Studierenden wurde mittels State-Trait-Angstinventar (STAI) in Ruhe und nach dem Anamnesegespräch gemessen. Die physische Stresskomponente wurde durch vier Parameter der Herzfrequenzvariabilität (HRV) (RMSSD, HF power, HF power (log), HF power (n.u.)) in Ruhe und während des Anamnesegesprächs erfasst. Die Anamnesegespräche verursachten bei den Medizinstudierenden im Vergleich zur Ruhesituation in den Gesprächsszenarien Stress (rpsych = .36 (p = .018) und rphys = .44 (p < .001)). Beim Vergleich des Stresslevels zwischen den drei Gesprächsszenarien, verhielten sich die psychische und die physische Stresskomponente bei zwei Szenarien entgegengesetzt: So zeigte sich im SP anhand des STAI signifikant mehr Stress als im EP (r = .33, p = .039). Das genau gegenteilige Bild zeigten die HRV Parameter; dort war EP mit signifikant mehr Stress verbunden als das SP (r = .28, p = .034). Die Erklärungsansätze hierfür sind mannigfaltig. Diese fundiert einzuordnen, ist jedoch aufgrund der limitierten Anzahl an Studien zu diesem Thema schwierig. Zwei vielversprechende Gründe könnten sein: (1.) Es gab eine zeitliche Verzögerung bei der Erhebung der psychischen Stresskomponente im EP. In dieser Zeit könnte das Stresslevel bereits wieder abgefallen sein. Und (2.) die positive Einschätzung im Kontakt mit echten Patient/-innen könnte zu einem Eustress geführt haben, welcher das subjektive Stresserleben günstig beeinflusste. Hier bedarf es weiterer Forschung um die Frage abschließend zu beantworten. Diesem Ergebnis entsprechend konnte kein signifikanter Zusammenhang zwischen der psychischen und der physischen Stresskomponente während des Anamnesegesprächs aufgezeigt werden. Diese Arbeit soll zur Qualitätsoptimierung der Lehre im Medizinstudium beitragen, namentlich der Einübung des Anamnesegesprächs. Auf Basis der gewonnen Erkenntnisse kann weitere Lehrforschung anschließen, um die Zusammenhänge von Stresslevel, Gesprächspartner/-in und Lernerfolg zu untersuchen. Davon würden sowohl die Medizinstudierenden als auch die Patient/-innen profitieren

Understanding the impact of relationship disruptions

Personal relationships between salespeople and customers are essential for the success of business-to-business relationships, and research has shown that a change of the salesperson can severely harm financial performance. However, such interpersonal relationship disruptions may also have positive effects by encouraging vitalizing reexplorations of the relationship. Using multilevel loyalty theory and relationship life cycle theory, the authors offer a comprehensive conceptualization of potentially countervailing consequences of relationship disruptions. In particular, disruptions may have different effects on resale revenue (from previously sold products) versus new sale revenue (from newly sold products), contingent on both the history and expected future development of the relationship. Therefore, this study examines moderators on the firm-level relationship prior to disruption and salesperson relationship management afterward. Longitudinal data from 2,040 customers of an international business-to-business firm reveal that a disruption can increase overall performance by more than 29%, depending on the firm-level relationship before disruption and the new salesperson’s relationship management. Managers can use these findings proactively to evaluate and manage the risks and opportunities involved in relationship disruptions

Learning Combinatorial Node Labeling Algorithms

We present a graph neural network to learn graph coloring heuristics using reinforcement learning. Our learned deterministic heuristics give better solutions than classical degree-based greedy heuristics and only take seconds to evaluate on graphs with tens of thousands of vertices. As our approach is based on policy-gradients, it also learns a probabilistic policy as well. These probabilistic policies outperform all greedy coloring baselines and a machine learning baseline. Our approach generalizes several previous machine-learning frameworks, which applied to problems like minimum vertex cover. We also demonstrate that our approach outperforms two greedy heuristics on minimum vertex cover

A Cross-Sectional, Abattoir-Based Study

Abstract Toxigenic Escherichia coli (E. coli) are an important cause of gastroenteritis in developing countries. In Ethiopia, gastroenteritis due to food-borne disease is a leading cause of death. Yet, there is no surveillance for E. coli O157 and little is known about the carriage of this pathogen in Ethiopia’s livestock. This study aimed to assess the prevalence and levels of antimicrobial resistance of E. coli O157 in goat meat, feces, and environmental samples collected at a large abattoir in the Somali region of Ethiopia. The samples were enriched in modified tryptone broth containing novobiocin, and plated onto sorbitol MacConkey agar. Isolates were confirmed using indole test and latex agglutination. Antimicrobial susceptibility testing was conducted using the disk diffusion method. A total of 235 samples, including 93 goat carcass swabs, 93 cecal contents, 14 water, 20 hand, and 15 knife swabs were collected. Overall, six (2.5%) samples were contaminated with E. coli O157 of which two (2.1%) were isolated from cecal contents, three (3.2%) from carcass swabs, and one (7.1%) from water. All isolates were resistant to at least two of the 18 antimicrobials tested. Two isolates (33.3%) were resistant to more than five antimicrobials. Abattoir facilities and slaughter techniques were conducive to carcass contamination. This study highlights how poor hygiene and slaughter practice can result in contaminated meat, which is especially risky in Ethiopia because of the common practice of eating raw meat. We detect multi-resistance to drugs not used in goats, suggesting that drugs used to treat human infections may be the originators of antimicrobial resistance in livestock in this ecosystem. The isolation of multidrug-resistant E. coli O157 from goats from a remote pastoralist system highlights the need for global action on regulating and monitoring antimicrobial use in both human and animal populations

Fast single-cell biochemistry: theory, open source microscopy and applications

Fluorescence lifetime sensing enables researchers to probe the physicochemical environment of a fluorophore providing a window through which we can observe the complex molecular make-up of the cell. Fluorescence lifetime imaging microscopy (FLIM) quantifies and maps cell biochemistry, a complex ensemble of dynamic processes. Unfortunately, typical high-resolution FLIM systems exhibit rather limited acquisition speeds, often insufficient to capture the time evolution of biochemical processes in living cells. Here, we describe the theoretical background that justifies the developments of high-speed single photon counting systems. We show that systems with low dead-times not only result in faster acquisition throughputs but also improved dynamic range and spatial resolution. We also share the implementation of hardware and software as an open platform, show applications of fast FLIM biochemical imaging on living cells and discuss strategies to balance precision and accuracy in FLIM. The recent innovations and commercialisation of fast time-domain FLIM systems are likely to popularise FLIM within the biomedical community, to impact biomedical research positively and to foster the adoption of other FLIM techniques as well. While supporting and indeed pursuing these developments, with this work we also aim to warn the community about the possible shortcomings of fast single photon counting techniques and to highlight strategies to acquire data of high quality.We acknowledge funding from the Medical Research Council core grants (MC_UU_12022/1 and MC_UU_12022/8) to ARV

Levantamento gravimétrico na Região de Franca - SP e Delfinópolis - MG, no reconhecimento e delimitação de feições estruturais (Zonas de Sutura)

This paper presents the results of a geophysical study carried out in northeastern São Paulo State and southwestern Minas Gerais State over an area 80 km wide and 97.5 km long in SE Brazil. The Bouguer anomaly map, and geological and structural data allowed to identify three different gravity domains - crustal blocks limited by major discontinuities -related to the structural pattern of the area. These discontinuities were interpreted as geosuture zones underlying the Paraná Basin sediments which have extensions in the Pre-cambrian Basement. The crustal discontinuities named Alterosa and Ribeirão Preto may be seen as A - type collision sutures in a triple junction arramgement. Two prominent linear anomalies are recognized in the Bouguer anomaly map, as well as the limit between the Brasília and São Paulo crustal blocks or paleoplates. The Alterosa suture zone trends NW-SE while the Ribeirão Preto suture has a NE-SW direction. The Bouguer anomaly map provides subsidies and information on new concepts and theories leading to the refinement of tectonic models.Este trabalho apresenta o resultado do levantamento geofísico realizado no nordeste do estado de São Paulo e sudoeste do estado de Minas Gerais em uma área de 80 por 97,5 km localizada a sudoeste do Brasil. O mapa da anomalia Bouguer associado a estudos geológicos e estruturais permite identificar basicamente três grandes domínios gravimétricos relacionados ao arranjo estrutural e compartimentação crustal (blocos crustais delimitados por grandes descontinuidades) na área. Essas descontinuidades foram interpretadas em estudos anteriores como zonas de geosuturas localizadas abaixo dos sedimentos da Bacia sedimentar do Paraná e possuem um prolongamento no embasamento Pré - Cambriano. As descontinuidades denominadas de Alterosa e Ribeirão Preto são classificadas como sendo suturas colisionais do tipo - A compondo um arranjo de junção tríplice. São reconhecidas no mapa da anomalia Bouguer duas anomalias lineares e os limites entre os blocos crustais ou paleo-placas Brasília e São Paulo. A zona de sutura de Alterosa possui uma direção preferencial NW-SE e na direção NE-SW encontra-se a zona de sutura de Ribeirão Preto. O mapa da anomalia Bouguer gerado fornece subsídios e informações para novos conceitos e teorias de modelos tectônicos propostos

PROSPECÇÃO GEOFÍSICA ENTRE AS OCORRÊNCIAS CUPRÍFERAS CAPÃO GRANDE E VICTOR TEIXEIRA, CAÇAPAVA DO SUL – RS

Esse trabalho consiste na aquisição de dados de magnetometria terrestre numa área compreendida por duas ocorrências cupríferas denominadas Capão Grande e Victor Teixeira, localizadas no interior do município de Caçapava do Sul-RS. Os dados resultantes da aplicação desse método geofísico foram adquiridos com o propósito de caracterizar a subsuperfície para verificar uma potencial relação de conexão entre as mineralizações dessas ocorrências de cobre. Capão Grande tem como encaixante as rochas sedimentares da Bacia do Camaquã. Victor Teixeira está inserida em rochas metamórficas do Escudo Sul-Rio-Grandense. Na caracterização de subsuperfície foram empregados levantamentos de medidas de intensidade do campo magnético total ao longo de perfis e sob a forma de malha no intervalo entre as ocorrências mineralizadas. Essas medidas foram processadas em conjunto, e os produtos resultantes foram os mapas do Campo Magnético Total, Passa Banda Profundo, Passa Banda Raso e Amplitude do Sinal Analítico. Análises e discussões posteriores desses mapas com a sobreposição das principais informações geológicas da área e de resultados geofísicos de interesse de estudos anteriores permitiram a definição de que as mineralizações das ocorrências estudadas não apresentam conexão em subsuperfície

Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two biochemical reactions in living cells are limited. Therefore, the development of methods for real-time biochemical multiplexing is of fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described with multidimensional phasor transforms. We demonstrate a computational framework capable of demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed biochemical activities in single living cells. We provide a comparison between mcFLIM and sFLIM suggesting that sFLIM might be advantageous for the future development of heavily multiplexed assays. However, mcFLIM—more readily available with commercial systems—can be applied for the concomitant monitoring of three enzymes in living cells without significant losses