308 research outputs found

    Applications of Molecular Dynamics simulations for biomolecular systems and improvements to density-based clustering in the analysis

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    Molecular Dynamics simulations provide a powerful tool to study biomolecular systems with atomistic detail. The key to better understand the function and behaviour of these molecules can often be found in their structural variability. Simulations can help to expose this information that is otherwise experimentally hard or impossible to attain. This work covers two application examples for which a sampling and a characterisation of the conformational ensemble could reveal the structural basis to answer a topical research question. For the fungal toxin phalloidin—a small bicyclic peptide—observed product ratios in different cyclisation reactions could be rationalised by assessing the conformational pre-organisation of precursor fragments. For the C-type lectin receptor langerin, conformational changes induced by different side-chain protonations could deliver an explanation of the pH-dependency in the protein’s calcium-binding. The investigations were accompanied by the continued development of a density-based clustering protocol into a respective software package, which is generally well applicable for the use case of extracting conformational states from Molecular Dynamics data

    k-Means

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    Engineering Proteins by Domain Insertion

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    Protein domains are structural and functional subunits of proteins. The recombination of existing domains is a source of evolutionary innovation, as it can result in new protein features and functions. Inspired by nature, protein engineering commonly uses domain recombination in order to create artificial proteins with tailor-made properties. Customized control over protein activity, for instance, can be achieved by harnessing switchable domains and functionally linking them to effector domains. Many natural protein domains exhibit conformational changes in response to exogenous triggers. The insertion of light-switchable receptor domains into an effector protein of choice, for instance, allows the control of effector activity with light. The resulting optogenetic proteins represent powerful tools for the investigation of dynamic cellular processes with high precision in time and space. On top, optogenetic proteins enable manifold biotechnological applications and they are even considered potential candidates for future therapeutics. In this study, we first focused on CRISPR-Cas9 genome editing and applied a domain insertion strategy to genetically encoded inhibitors of the CRISPR nuclease from Neisseria meningitidis (NmeCas9), which due to its small size and high DNA sequence-specificity is of great interest for CRISPR genome editing applications. Fusing stabilizing domains to the NmeCas9 inhibitory protein AcrIIC1 allowed us to boost its inhibitory effect, thereby yielding a potent gene editing off-switch. Furthermore, the insertion of the light-responsive LOV2 domain from Avena sativa into AcrIIC3, the most potent inhibitor of NmeCas9, enabled the optogenetic control of gene editing via light-dependent NmeCas9 inhibition. Further investigation of the engineered inhibitors revealed the potential these proteins could have with respect to safe-guarding of the CRISPR technology by selectively reducing off-target editing. The laborious optimization of the engineered CRISPR inhibitors necessary by the time motivated us to more systematically investigate possibilities and constraints of protein engineering by domain insertion using an unbiased insertion approach. Previously, single protein domains were usually introduced only at a few rationally selected sites into target proteins. Here, we inserted up to five structurally and functionally unrelated domains into several different candidate effector proteins at all possible positions. The resulting libraries of protein hybrids were screened for activity by fluorescence-activated cell sorting (FACS) and subsequent next-generation sequencing (Flow-seq). Training machine learning models on the resulting, comprehensive datasets allowed us to dissect parameters that affect domain insertion tolerance and revealed that sequence conservation statistics are the most powerful predictors for domain insertion success. Finally, extending our experimental Flow-seq pipeline towards the screening of engineered, switchable effector variants yielded two potent optogenetic derivatives of the E. coli transcription factor AraC. These novel hybrids will enable the co-regulation of bacterial gene expression by light and chemicals. Taken together, our study showcases the design of functionally diverse protein switches for the control of gene editing and gene expression in mammalian cells and E. coli, respectively. In addition, the generation of a large domain insertion datasets enabled - for the first time - the unbiased investigation of domain insertion tolerance in several evolutionary unrelated proteins. Our study showcases the manifold opportunities and remaining challenges behind the engineering of proteins with new properties and functionalities by domain recombination

    The universe without us: a history of the science and ethics of human extinction

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    This dissertation consists of two parts. Part I is an intellectual history of thinking about human extinction (mostly) within the Western tradition. When did our forebears first imagine humanity ceasing to exist? Have people always believed that human extinction is a real possibility, or were some convinced that this could never happen? How has our thinking about extinction evolved over time? Why do so many notable figures today believe that the probability of extinction this century is higher than ever before in our 300,000-year history on Earth? Exploring these questions takes readers from the ancient Greeks, Persians, and Egyptians, through the 18th-century Enlightenment, past scientific breakthroughs of the 19th century like thermodynamics and evolutionary theory, up to the Atomic Age, the rise of modern environmentalism in the 1970s, and contemporary fears about climate change, global pandemics, and artificial general intelligence (AGI). Part II is a history of Western thinking about the ethical and evaluative implications of human extinction. Would causing or allowing our extinction be morally right or wrong? Would our extinction be good or bad, better or worse compared to continuing to exist? For what reasons? Under which conditions? Do we have a moral obligation to create future people? Would past “progress” be rendered meaningless if humanity were to die out? Does the fact that we might be unique in the universe—the only “rational” and “moral” creatures—give us extra reason to ensure our survival? I place these questions under the umbrella of Existential Ethics, tracing the development of this field from the early 1700s through Mary Shelley’s 1826 novel The Last Man, the gloomy German pessimists of the latter 19th century, and post-World War II reflections on nuclear “omnicide,” up to current-day thinkers associated with “longtermism” and “antinatalism.” In the dissertation, I call the first history “History #1” and the second “History #2.” A main thesis of Part I is that Western thinking about human extinction can be segmented into five distinction periods, each of which corresponds to a unique “existential mood.” An existential mood arises from a particular set of answers to fundamental questions about the possibility, probability, etiology, and so on, of human extinction. I claim that the idea of human extinction first appeared among the ancient Greeks, but was eclipsed for roughly 1,500 years with the rise of Christianity. A central contention of Part II is that philosophers have thus far conflated six distinct types of “human extinction,” each of which has its own unique ethical and evaluative implications. I further contend that it is crucial to distinguish between the process or event of Going Extinct and the state or condition of Being Extinct, which one should see as orthogonal to the six types of extinction that I delineate. My aim with the second part of the book is to not only trace the history of Western thinking about the ethics of annihilation, but lay the theoretical groundwork for future research on the topic. I then outline my own views within “Existential Ethics,” which combine ideas and positions to yield a novel account of the conditions under which our extinction would be bad, and why there is a sense in which Being Extinct might be better than Being Extant, or continuing to exist

    LIPIcs, Volume 277, GIScience 2023, Complete Volume

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    LIPIcs, Volume 277, GIScience 2023, Complete Volum

    12th International Conference on Geographic Information Science: GIScience 2023, September 12–15, 2023, Leeds, UK

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    No abstract available

    2023-2024 Lynn University Academic Catalog

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    The 2023-2024 Academic Catalog initially published as a web-only document. The Department of Marketing and Communication created a PDF version, which is available for download here.https://spiral.lynn.edu/accatalogs/1052/thumbnail.jp
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