No description available.

DNA bending plays a vital role in numerous cellular activities such as transcription, viral packaging, and nucleosome formation. Therefore, understanding the physics of DNA bending at the length scales relevant to these processes is one of the main keys to the quantitative description of life. However, previous studies provide a divided picture on how DNA should be modeled in strong bending condition relevant to biology. My thesis is devoted to answering how far an elastic rod model can be applied to DNA. We consider several subtle features that could potentially lead to the break-down of the worm-like chain model, such as local bendedness of the sequence and large bending angles. We used single-molecule Fluorescence Resonance Energy Transfer to track looping and unlooping of single DNA molecules in real time. We compared the measured looping and unlooping rates with theoretical predictions of the worm-like chain model. We found that the intrinsic curvature of the sequence affects the looping propensity of short DNA and an extended worm-like chain model including the helical parameters of individual base pairs could adequately explain our measurements. For DNA with random sequence and negligible curvature, we discovered that the worm-like chain model could explain the stability of small DNA loops only down to a critical loop size. Below the critical loop size, the bending stress stored in the DNA loop became less sensitive to loop size, indicative of softened dsDNA. The critical loop size is sensitive to salt condition, especially to magnesium at mM concentrations. This finding enabled us to explain several contrasting results in the past and shed new light on the energetics of DNA bending.

Telomerase and telomere play crucial roles in the maintenance of genomic stability. Through its ability to extend chromosome ends with G-rich telomeric sequence, telomerase solves the end-replication problem of linear chromosomes and allows complete replication of the genetic information. Telomere along with its protein partners solves the end-protection problem and guards the chromosome ends against aberrant DNA damage response. In this thesis, I present two single-molecule fluorescence-based studies that determined the functional structure of telomerase RNA within active telomerase holoenzyme and probed the structure of telomere and its dependence on telomere binding proteins. In the first study, we developed a single-molecule Förster resonance energy transfer (FRET) assay to interrogate the structure of telomerase RNA within active telomerase enzymes. In this assay, oligonucleotide hybridization was used to probe the primer-extension activity of individual telomerase enzymes with single nucleotide sensitivity. FRET signals from individual enzyme molecules during active binding events were then used to determine the organization of telomerase RNA within active telomerase. Using this assay, we have identified an active conformation of telomerase in which the conserved telomerase RNA pseudoknot is properly folded. In the second study, we used super-resolution fluorescence technique STochastic Optical Reconstruction Microscopy (STORM) to probe the structure of mammalian telomere. We showed that previously described telomere loop structures are detected by STORM imaging. Removal of telomere-binding protein TRF2 significantly reduces the fraction of telomeres found in loops. Furthermore, this reduction of telomere loops occurs in the absence of ATM-dependent DNA damage signaling and non-homologous end joining mediated chromosome fusion, suggesting a direct role of TRF2 in the formation or maintenance of telomere loops.

No description available.

No description available.

Alzheimer's disease (AD) is the most common form of dementia among elderly. The disease is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles, loss of synapses and neurons and chronic neuroinflammation. The significance of neuroinflammatory processes in disease on-set and progression has been debated since activated microglia and reactive astrocytes have been attributed both protective and damaging properties. However, patients systematically treated with anti-inflammatory drugs have been shown to develop AD to a lesser extent than average. This indicates an important role of neuroinflammation in AD. This thesis focuses on two inflammatory related transcription factors, nuclear factor κB (NF-κB) and CCAAT/enhancer binding protein (C/EBP). Both NF-κB and C/EBP are known regulators of many pro-inflammatory genes and may during certain circumstances dimerize with each other. In paper I we use a new strategy to inhibit NF-κB DNA binding activity in primary astro-microglial cell cultures treated with Aβ and IL-1β. By coupling the NF-κB decoy to a transport peptide both concentration and incubation time can be shortened in comparison to previous studies. Moreover, using the same in vitro model in paper II and III, we show members of the C/EBP family to be dysregulated during AD mimicking conditions. Additional focus was directed towards C/EBPδ, which was shown to respond differently to oligomeric and fibrillar forms of Aβ. Results were also confirmed in vivo using an AD mouse model characterized by high levels of fibrillar Aβ deposits. Finally, in order to get further insight in neurodegenerative processes, induced by Aβ or microglial activation, we present in paper IV a new set of anchored sensors for detection of locally activated caspases in neuronal cells. By anchoring the sensors to tau they become less dynamic and caspase activation can be detected early on in the apoptotic process, in a spatio-temporal and reproducible manner.

The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.

Fluorescence single-molecule methods represent mighty tools for researchers in the field of structural and molecular biology. These methods are bringing in many advantages when compared to the statistical data processing of multi-molecular species. We can directly compare true statistical distributions and their kinetics. Here belongs fluorescence correlation spectroscopy, FRET and burst variance analysis. As the research advances, new methods are being developed which at the very beginning do not have proper analytical relations for data interpretation and which experimental limits we can't tell. That is the moment when the computer simulations can be used to our advantage. They can help us to specify the right direction of the future research, which is a great money-saver, while opening new perspectives and insights into the explored matter. Correct interpretation of the simulations results is key for the consequent defining of new theoretical models.

En général, cette recherche se compose de quatre parties, sur fond de recherche , l'identification des risques, évaluation des risques et des suggestions de contrôle des risques. Arrière-plan de recherche comprend l'introduction de la recherche, l'auteur fond et revue de la littérature sur les théories et les concepts clés et les mécanismes de formulation de risque. L'identification des risques est la première étape importante de la gestion du risque d'entreprise. Dans cette recherche, remue-méninges, une interview, RBS et les méthodes de revue de la littérature seront utilisés ensemble pour identifier les risques dans l'industrie chinoise de fret aérien. Du point de vue de la théorie, il expliquera pourquoi il choisit ces méthodes et met en oeuvre la comparaison des méthodes. Grâce à l'interview, enquête auprès des experts, deux couches de facteurs de risque ont été créées. Pour les méthodes d'évaluation des risques, il y a principalement environ 14 méthodes indépendantes d'évaluation des risques sont discutés, veille stratégique, l'analyse du pire cas et ainsi de suite. Pour l'évaluation de l'industrie chinoise de fret aérien du risque, l'ANP est une méthode scientifique, raisonnable et pratique. Le jugement et la suggestion des experts sont indispensables et complémentaires dans l'évaluation des risques, y compris la méthode de l'ANP. En raison de la pratique de l'identification des risques, l'auteur a une relation étroite avec la plupart des experts au sein de l'industrie chinoise de fret aérien. 20 experts ont été choisis comme les personnes interrogées. Basé sur les 17 questionnaires valides et des logiciels de Superdecision, le processus d'évaluation des risques réalisée toutes les priorités pour tous les facteurs de risque. Selon le résultat, 19 risques sont classés en trois catégories, les risques rouge, jaune et blanc. Pour chaque type de risques, tous les participants peuvent prendre des décisions contre des risques en fonction du résultat de l'évaluation. L'évaluation des risques n'est pas la fin de la gestion des risques pour l'industrie chinoise de fret aérien, le but de l'évaluation des risques est de soutenir la réalisation et apporter des inférences décision. En Chine, le eCargo est une pratique de ACSCEP qui est proposé par le gouvernement de la Chine. En tout, ACSCEP peut résoudre de nombreux risques de fret aérien chinois. En tant qu'expert et membre de la plateforme eCargo, j'ai proposé que eCargo est un bon des contre-mesures de risque. / Generally this research consists of four parts, research background, risk identification, risk assessment and risk control suggestions. Research background consists of research introduction, author background and literature reviews on key concepts and theories, and risk formulation mechanisms. Risk identification is the first and important step of enterprise risk management. In this research, brainstorm, expert interview, RBS and literature review methods will be used together to identify the risk in Chinese air cargo industry. From theory perspective, it will explain why it chooses these methods and implements the methods comparison. Through the interview, survey with experts, two layers of risk factors have been founded. For the risk assessment methods, there are mainly about 14 independent risk assessment methods are discussed, as strategic scanning, worst-case analysis and so on. For the risk assessment of Chinese air cargo industry, ANP is a scientific, reasonable and practical method. Experts’ judgment and suggestion are indispensable and complementary in risk assessment, including ANP method. Because of the practice of risk identification, the author has a close relationship with most of the experts within Chinese air cargo industry. 20 experts were chosen as the interviewees. Based on the 17 valid questionnaires and Superdecision software, risk assessment process produced all priorities for all the risk factors. According the result, 19 risks are classified into three categories, red, yellow and white risks. For each kind of risks, all the participants can make risk counter decisions according to the assessment result. Risk assessment is not the end for risk management for Chinese air cargo industry, the purpose of the risk assessment is to support the decision making and bring inferences. In China, the Ecargo is one practice of ACSCEP which is proposed by China government. In all, ACSCEP can solve many Chinese air cargo risks. As an expert and member of Ecargo platform, I proposed that Ecargo is a good risk counter measures.

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.