Self-replicating Replicon-RNA Delivery to Dendritic Cells by Chitosan-nanoparticles for Translation In Vitro and In Vivo.

Self-amplifying replicon RNA (RepRNA) possesses high potential for increasing antigen load within dendritic cells (DCs). The major aim of the present work was to define how RepRNA delivered by biodegradable, chitosan-based nanoparticulate delivery vehicles (nanogel-alginate (NGA)) interacts with DCs, and whether this could lead to translation of the RepRNA in the DCs. Although studies employed virus replicon particles (VRPs), there are no reports on biodegradable, nanoparticulate vehicle delivery of RepRNA. VRP studies employed cytopathogenic agents, contrary to DC requirements-slow processing and antigen retention. We employed noncytopathogenic RepRNA with NGA, demonstrating for the first time the efficiency of RepRNA association with nanoparticles, NGA delivery to DCs, and RepRNA internalization by DCs. RepRNA accumulated in vesicular structures, with patterns typifying cytosolic release. This promoted RepRNA translation, in vitro and in vivo. Delivery and translation were RepRNA concentration-dependent, occurring in a kinetic manner. Including cationic lipids with chitosan during nanoparticle formation enhanced delivery and translation kinetics, but was not required for translation of immunogenic levels in vivo. This work describes for the first time the characteristics associated with chitosan-nanoparticle delivery of self-amplifying RepRNA to DCs, leading to translation of encoded foreign genes, namely influenza virus hemagglutinin and nucleoprotein.

Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines

Self-amplifying replicon RNA (RepRNA) are large molecules (12-14kb); their self-replication amplifies mRNA template numbers, affording several rounds of antigen production, effectively increasing vaccine antigen payloads. Their sensitivity to RNase-sensitivity and inefficient uptake by dendritic cells (DCs) - absolute requirements for vaccine design - were tackled by condensing RepRNA into synthetic, nanoparticulate, polyethylenimine (PEI)-polyplex delivery vehicles. Polyplex-delivery formulations for small RNA molecules cannot be transferred to RepRNA due to its greater size and complexity; the N:P charge ratio and impact of RepRNA folding would influence polyplex condensation, post-delivery decompaction and the cytosolic release essential for RepRNA translation. Polyplex-formulations proved successful for delivery of RepRNA encoding influenza virus hemagglutinin and nucleocapsid to DCs. Cytosolic translocation was facilitated, leading to RepRNA translation. This efficacy was confirmed in vivo, inducing both humoral and cellular immune responses. Accordingly, this paper describes the first PEI-polyplexes providing efficient delivery of the complex and large, self-amplifying RepRNA vaccines.

Tilings induced by a class of cubic Rauzy fractals

We study aperiodic and periodic tilings induced by the Rauzy fractal and its subtiles associated with beta-substitutions related to the polynomial x3-ax2-bx-1 for a≥b≥1. In particular, we compute the corresponding boundary graphs, describing the adjacencies in the tilings. These graphs are a valuable tool for more advanced studies of the topological properties of the Rauzy fractals. As an example, we show that the Rauzy fractals are not homeomorphic to a closed disc as soon as a≤2b-4. The methods presented in this paper may be used to obtain similar results for other classes of substitutions.© 2012 Elsevier B.V. All rights reserved.

Directed evolution of polymerase function by compartmentalized self-replication

We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.

Inventing life: Patent law and synthetic biology

With promises of improved medical treatments, greener energy and even artificial life, the field of synthetic biology has captured the public imagination and attracted significant government and commercial investment. This excitement reached a crescendo on 21 May 2010, when scientists at the J Craig Venter Institute in the United States announced that they had made a “self-replicating synthetic bacterial cell”. This was the first living cell to have an entirely human-made genome, which means that all of the cell’s characteristics were controlled by a DNA sequence designed by scientists. This achievement in biological engineering was made possible by combining molecular biotechnology, gene synthesis technology and information technology.

Language change as an evolutionary process

In the thesis it is discussed in what ways concepts and methodology developed in evolutionary biology can be applied to the explanation and research of language change. The parallel nature of the mechanisms of biological evolution and language change is explored along with the history of the exchange of ideas between these two disciplines. Against this background computational methods developed in evolutionary biology are taken into consideration in terms of their applicability to the study of historical relationships between languages. Different phylogenetic methods are explained in common terminology, avoiding the technical language of statistics. The thesis is on one hand a synthesis of earlier scientific discussion, and on the other an attempt to map out the problems of earlier approaches in addition to finding new guidelines in the study of language change on their basis. Primarily literature about the connections between evolutionary biology and language change, along with research articles describing applications of phylogenetic methods into language change have been used as source material. The thesis starts out by describing the initial development of the disciplines of evolutionary biology and historical linguistics, a process which right from the beginning can be seen to have involved an exchange of ideas concerning the mechanisms of language change and biological evolution. The historical discussion lays the foundation for the handling of the generalised account of selection developed during the recent few decades. This account is aimed for creating a theoretical framework capable of explaining both biological evolution and cultural change as selection processes acting on self-replicating entities. This thesis focusses on the capacity of the generalised account of selection to describe language change as a process of this kind. In biology, the mechanisms of evolution are seen to form populations of genetically related organisms through time. One of the central questions explored in this thesis is whether selection theory makes it possible to picture languages are forming populations of a similar kind, and what a perspective like this can offer to the understanding of language in general. In historical linguistics, the comparative method and other, complementing methods have been traditionally used to study the development of languages from a common ancestral language. Computational, quantitative methods have not become widely used as part of the central methodology of historical linguistics. After the fading of a limited popularity enjoyed by the lexicostatistical method since the 1950s, only in the recent years have also the computational methods of phylogenetic inference used in evolutionary biology been applied to the study of early language history. In this thesis the possibilities offered by the traditional methodology of historical linguistics and the new phylogenetic methods are compared. The methods are approached through the ways in which they have been applied to the Indo-European languages, which is the most thoroughly investigated language family using both the traditional and the phylogenetic methods. The problems of these applications along with the optimal form of the linguistic data used in these methods are explored in the thesis. The mechanisms of biological evolution are seen in the thesis as parallel in a limited sense to the mechanisms of language change, however sufficiently so that the development of a generalised account of selection is deemed as possibly fruiful for understanding language change. These similarities are also seen to support the validity of using phylogenetic methods in the study of language history, although the use of linguistic data and the models of language change employed by these models are seen to await further development.

Implications of streamlining theory for microbial ecology

Whether a small cell, a small genome or a minimal set of chemical reactions with self-replicating properties, simplicity is beguiling. As Leonardo da Vinci reportedly said, 'simplicity is the ultimate sophistication'. Two diverging views of simplicity have emerged in accounts of symbiotic and commensal bacteria and cosmopolitan free-living bacteria with small genomes. The small genomes of obligate insect endosymbionts have been attributed to genetic drift caused by small effective population sizes (Ne). In contrast, streamlining theory attributes small cells and genomes to selection for efficient use of nutrients in populations where Ne is large and nutrients limit growth. Regardless of the cause of genome reduction, lost coding potential eventually dictates loss of function. Consequences of reductive evolution in streamlined organisms include atypical patterns of prototrophy and the absence of common regulatory systems, which have been linked to difficulty in culturing these cells. Recent evidence from metagenomics suggests that streamlining is commonplace, may broadly explain the phenomenon of the uncultured microbial majority, and might also explain the highly interdependent (connected) behavior of many microbial ecosystems. Streamlining theory is belied by the observation that many successful bacteria are large cells with complex genomes. To fully appreciate streamlining, we must look to the life histories and adaptive strategies of cells, which impose minimum requirements for complexity that vary with niche.

Viral marketing and the new competitive advantage

The role played by viral marketing has received considerable academic and digital media attention recently. Key issues in viral marketing have been examined through the lens of the mode of marketing message transmission, including self-replicating on the basis of quality difference, individuals’ emotional needs, as well as how users are connected across various social networks. This paper presents a review and analysis of viral marketing studies from 2001 to the present day. It investigates how viral marketing facilitate the diffusion of social media products and the relationship between marketers and these product users by taking a look at the implementation of viral marketing in two European online game firms Jagex Games Studio and Rovio Entertainment. The results from this review and analysis indicate that viral marketing plays an important role in accelerating the interaction between marketers and users (as well as the user groups) in the field of digital media and high tech consumption. Therefore, it is evident that firms should understand the social contagion process and target well-connected users purposefully in order to create its competitive advantage.

Solvable model for template coexistence in protocells

Compartmentalization of self-replicating molecules (templates) in protocells is a necessary step towards the evolution of modern cells. However, coexistence between distinct template types inside a protocell can be achieved only if there is a selective pressure favoring protocells with a mixed template composition. Here we study analytically a group selection model for the coexistence between two template types using the diffusion approximation of population genetics. The model combines competition at the template and protocell levels as well as genetic drift inside protocells. At the steady state, we find a continuous phase transition separating the coexistence and segregation regimes, with the order parameter vanishing linearly with the distance to the critical point. In addition, we derive explicit analytical expressions for the critical steadystate probability density of protocell compositions.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

THE USE OF LIFE-CYCLE ANALYSIS TO REDUCE THE ENVIRONMENTAL IMPACT OF MATERIALS IN MANUFACTURING

This thesis is composed of three life-cycle analysis (LCA) studies of manufacturing to determine cumulative energy demand (CED) and greenhouse gas emissions (GHG). The methods proposed could reduce the environmental impact by reducing the CED in three manufacturing processes. First, industrial symbiosis is proposed and a LCA is performed on both conventional 1 GW-scaled hydrogenated amorphous silicon (a-Si:H)-based single junction and a-Si:H/microcrystalline-Si:H tandem cell solar PV manufacturing plants and such plants coupled to silane recycling plants. Using a recycling process that results in a silane loss of only 17 versus 85 percent, this results in a CED savings of 81,700 GJ and 290,000 GJ per year for single and tandem junction plants, respectively. This recycling process reduces the cost of raw silane by 68 percent, or approximately $22.6 and $79 million per year for a single and tandem 1 GW PV production facility, respectively. The results show environmental benefits of silane recycling centered around a-Si:H-based PV manufacturing plants. Second, an open-source self-replicating rapid prototype or 3-D printer, the RepRap, has the potential to reduce the environmental impact of manufacturing of polymer-based products, using distributed manufacturing paradigm, which is further minimized by the use of PV and improvements in PV manufacturing. Using 3-D printers for manufacturing provides the ability to ultra-customize products and to change fill composition, which increases material efficiency. An LCA was performed on three polymer-based products to determine the CED and GHG from conventional large-scale production and are compared to experimental measurements on a RepRap producing identical products with ABS and PLA. The results of this LCA study indicate that the CED of manufacturing polymer products can possibly be reduced using distributed manufacturing with existing 3-D printers under 89% fill and reduced even further with a solar photovoltaic system. The results indicate that the ability of RepRaps to vary fill has the potential to diminish environmental impact on many products. Third, one additional way to improve the environmental performance of this distributed manufacturing system is to create the polymer filament feedstock for 3-D printers using post-consumer plastic bottles. An LCA was performed on the recycling of high density polyethylene (HDPE) using the RecycleBot. The results of the LCA showed that distributed recycling has a lower CED than the best-case scenario used for centralized recycling. If this process is applied to the HDPE currently recycled in the U.S., more than 100 million MJ of energy could be conserved per annum along with significant reductions in GHG. This presents a novel path to a future of distributed manufacturing suited for both the developed and developing world with reduced environmental impact. From improving manufacturing in the photovoltaic industry with the use of recycling to recycling and manufacturing plastic products within our own homes, each step reduces the impact on the environment. The three coupled projects presented here show a clear potential to reduce the environmental impact of manufacturing and other processes by implementing complimenting systems, which have environmental benefits of their own in order to achieve a compounding effect of reduced CED and GHG.

Construcción de una maquina de control numérico " CNC" para prototipado rápido de piezas 3D en diferentes polímeros "impresora 3D"

El objetivo principal del presente Proyecto Fin de Carrera es la construcción , montaje y calibración de una impresora 3D auto replicable modelo Prusa Mendel capaz de trabajar en coordenadas polares, lo cual abre las puertas a la investigación de calidades, tolerancias, resistencias estructurales… de estas piezas en comparación con las fabricadas por impresoras cartesianas. Encontraras una guía de montaje paso a paso, además de un listado de todos los componentes, imprimibles y no imprimibles, que componen la impresora 3D. También se analizan y comparan las opciones a la hora de introducir la electrónica necesaria, extrusor y de los posibles errores y soluciones que se pueden encontrar durante la fabricación de una de estas máquinas. Finalmente dispondrás de una guía de calibración de skeinforce 41,para poder conseguir una impresión de gran calidad. Abstract The main objective of this Thesis is the construction, installation and calibration of a self-replicating 3D printer model Prusa Mendel able to work in polar coordinates, which opens the door to research quality, tolerances, these structural resistance ... parts compared to those manufactured by Cartesian printers. In this project you will find a guide step by step assembly, and a list of all components, and 3D printer components printable and unprintable. We also analyze and compare the options when entering the necessary electronics, extruder and possible errors and solutions that may occur during manufacturing of these machines finally have an installation guide calibration skeinforge 41 to get a high quality print

Guanidine hydrochloride blocks a critical step in the propagation of the prion-like determinant [PSI+] of Saccharomyces cerevisiae

The cytoplasmic heritable determinant [PSI+] of the yeast Saccharomyces cerevisiae reflects the prion-like properties of the chromosome-encoded protein Sup35p. This protein is known to be an essential eukaryote polypeptide release factor, namely eRF3. In a [PSI+] background, the prion conformer of Sup35p forms large oligomers, which results in the intracellular depletion of functional release factor and hence inefficient translation termination. We have investigated the process by which the [PSI+] determinant can be efficiently eliminated from strains, by growth in the presence of the protein denaturant guanidine hydrochloride (GuHCl). Strains are “cured” of [PSI+] by millimolar concentrations of GuHCl, well below that normally required for protein denaturation. Here we provide evidence indicating that the elimination of the [PSI+] determinant is not derived from the direct dissolution of self-replicating [PSI+] seeds by GuHCl. Although GuHCl does elicit a moderate stress response, the elimination of [PSI+] is not enhanced by stress, and furthermore, exhibits an absolute requirement for continued cell division. We propose that GuHCl inhibits a critical event in the propagation of the prion conformer and demonstrate that the kinetics of curing by GuHCl fit a random segregation model whereby the heritable [PSI+] element is diluted from a culture, after the total inhibition of prion replication by GuHCl.

Recombinant Kunjin virus replicon vaccines induce protective T-cell immunity against human papillomavirus 16 E7-expressing tumour

The persistence of the E7 oncoprotein in transformed cells in human papillomavirus (HPV)-associated cervical cancer provides a tumour-specific antigen to which immunotherapeutic strategies may be directed. Self-replicating RNA (replicon) vaccine vectors derived from the flavivirus Kunjin (KUN) have recently been reported to induce T-cell immunity. Here, we report that inclusion of a CTL epitope of HPV16 E7 protein into a polyepitope encoded by a KUN vector induced E7-directed T-cell responses and protected mice against challenge with an E7-expressing epithelial tumour. We found replicon RNA packaged into virus-like particles to be more effective than naked replicon RNA or plasmid DNA constructed to allow replicon RNA transcription in vivo. Protective immunity was induced although the E7 CTL epitope was subdominant in the context of other CTL epitopes in the polyepitope. The results demonstrate the efficacy of the KUN replicon vector system for inducing protective immunity directed towards a virally encoded human tumour-specific antigen, and for inducing multi-epitopic CTL responses. (C) 2004 Elsevier Inc. All rights reserved.

Reflecting on ethical and legal issues in wildlife disease

Disease in wildlife raises a number of issues that have not been widely considered in the bioethical literature. However, wildlife disease has major implications for human welfare. The majority of emerging human infectious diseases are zoonotic: that is, they occur in humans by cross-species transmission from animal hosts. Managing these diseases often involves balancing concerns with human health against animal welfare and conservation concerns. Many infectious diseases of domestic animals are shared with wild animals, although it is often unclear whether the infection spills over from wild animals to domestic animals or vice versa. Culling is the standard means of managing such diseases, bringing economic considerations, animal welfare and conservation into conflict. Infectious diseases are also major threatening processes in conservation biology and their appropriate management by culling, vaccination or treatment raises substantial animal ethics issues. One particular issue of great significance in Australia is an ongoing research program to develop genetically modified pathogens to control vertebrate pests including rabbits, foxes and house mice. Release of any self-replicating GMO vertebrate pathogen gives rise to a whole series of ethical questions. We briefly review current Australian legal responses to these problems. Finally, we present two unresolved problems of general importance that are exemplified by wildlife disease. First, to what extent can or should 'bioethics' be broadened beyond direct concerns with human welfare to animal welfare and environmental welfare? Second, how should the irreducible uncertainty of ecological systems be accounted for in ethical decision making?