1,720,956 research outputs found
Genetic controllers address noise-associated limitations of synthetic gene networks in E. coli. From the synchronization of genetic clocks to lysis-driven drug delivery
No full textThis thesis focuses on the design, building and testing of controllers for synthetic gene networks in bacterium E. coli, to address noise-associated limitations of such systems. Synthetic gene networks, also known as genetic circuits, can be implemented into bacterial cells in the form of genetic material (usually circular DNA molecules) programming the gene expression and behavior of host cells. Applications of genetic circuits in bacteria range from energy and metabolite production to the manipulation of microbial consortia and cancer therapy. However, stochasticity at both the single- cell and the population level affects the reliability of synthetic gene networks, sometimes hindering the purpose they are designed for. In this thesis, we applied rational bioengineering principles to integrate into underlying synthetic networks additional genetic parts, acting as designed external controllers. The controllers aimed at reducing the impact of noise in two genetic circuits in E. coli, with applications in the fields of non-linear physics and cancer therapy, respectively.
The first system is the repressilator, a renowned genetic circuit first presented in a seminal paper from 2000. It demonstrated the first experimental realization of a synthetic gene network designed from the bottom-up to produce periodic patterns of gene expression in individual bacteria. However, phase drifts and loss of synchronization are well-known noise-associated limitations of the repressilator, caused by the stochasticity of gene expression. We addressed these limitations by integrating an optogenetic module enabling to reset, delay, or advance the phase of the underlying oscillator using optical inputs. Our optically controllable genetic clock was the first experimental realization of a synthetic gene network to achieve long-term globally synchronized states through entrainment with a light cycle, with a mechanism reminiscent of natural circadian clocks. The high spatiotemporal resolution of optical stimulation and the simple structure of our network make it an optimal system to further investigate the non-linear dynamics of entrainment, phase adjustment and detuning in oscillating transcriptional networks.
The second system I worked on falls within the field of engineered bacteria cancer therapy. The field aims at exploiting genetic circuits to maximize the potential of bacteria to deliver therapeutic payloads directly to tumors, with precise control over therapeutic release in space and time. We characterized non- pathogenic E. coli expressing the bacterial toxin Perfringolysin O (PFO), a potent cancer cell cytotoxin, and presented experimental evidence that expression of PFO causes lysis of bacteria in both batch culture and microfluidic systems, facilitating its efficient release. However, we demonstrated that a major source of noise hindering the efficacy of the therapy is the emergence of a non-lysing mutant population that limits therapeutic-releasing bacteria in a PFO-expression level dependent manner. We addressed this limitation by integrating a chemically controllable genetic module enabling to regulate the rate and duration of pfo expression. We developed a mathematical model describing the evolution of therapeutic-releasing and mutant bacteria populations revealing trade-offs between therapeutic load delivered and fraction of mutants that arise. Altogether, we demonstrated how dynamic modulation of gene expression through external controllers can address mutant takeovers giving rise to limitations in engineered bacteria for therapeutic applications.
The two systems described above resulted in published papers on the journals eLife and SPJ BioDesign Research, respectively. In the last part of the thesis, we discuss the future perspectives of this work and present the design and characteriza- tion of additional controllers for lysis in E. coli. Notably, we describe an optogenetic controller that allows the fine-tuning of lysis in time and space, in response to three orthogonal wavelengths of light. We will end the thesis by addressing another question that the reader of this thesis might have: how can bacterial therapies evolve, going from bench to bedside? We will suggest an answer to this question by reviewing further developments of bacterial therapies: on the methodological side, reporting the construction of a microfluidic platform for the co-culture of engineered bacteria with human cancer cells, to evaluate the pharmacokinetics of bacterial therapies in vitro; and on the applicative side, introducing bacteria-based immunotherapies for cancer, another promising class of therapeutics whose efficacy could be enhanced by designed external controllers programming dynamic expression of the therapeutics.
Altogether, this thesis demonstrates that rationally-designed genetic controllers are an effective tool to counteract the deleterious impact of noise affecting synthetic gene networks in E. coli, which hinders the development of safer, more reliable and reproducible gene-based systems for biomedical applications and beyond
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Light-driven synchronization of optogenetic clocks
No full textSynthetic genetic oscillators can serve as internal clocks within engineered cells to program periodic expression. However, cell-to-cell variability introduces a dispersion in the characteristics of these clocks that drives the population to complete desynchronization. Here, we introduce the optorepressilator, an optically controllable genetic clock that combines the repressilator, a three-node synthetic network in E. coli, with an optogenetic module enabling to reset, delay, or advance its phase using optical inputs. We demonstrate that a population of optorepressilators can be synchronized by transient green light exposure or entrained to oscillate indefinitely by a train of short pulses, through a mechanism reminiscent of natural circadian clocks. Furthermore, we investigate the system’s response to detuned external stimuli observing multiple regimes of global synchronization. Integrating experiments and mathematical modeling, we show that the entrainment mechanism is robust and can be understood quantitatively from single cell to population level
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
- …
