A rational approach to biotransformation process design: Chemo-enzymatic synthesis of N-acetyl-D-neuraminic acid

This thesis describes a rational approach to process design for biotransformation processes. The approach enabled process flow-sheet decisions to be made based on structured scientific evidence and provided key data affecting design choices. The two step synthesis of N-acetyl-D-neuraminic acid (Neu5Ac) was taken as a model for process design. Neu5Ac was synthesised starting from N-acetyl-D-glucosamine (GlcNAc) and pyruvate (Pyr) in two steps. The first was the base-catalysed epimerisation of GlcNAc to N-acetyl-D-mannosamine (ManNAc). The second step was a biotransformation, carried out by the addition of pyruvate to ManNAc to yield Neu5Ac in equilibrium. The latter reaction was catalysed by the E. coli Neu5Ac aldolase. The first phase to the structured approach to biotransformation process design was based on characterisation. A first group of experiments investigated the components and reaction properties and this set of data indicated advantageous conditions on which to focus subsequent characterisation. The second set of data concerned the interactions between reactants, product and biocatalyst at the conditions selected above. Reaction kinetics at high substrate concentrations has determined that pyruvate was strongly inhibitory on initial rates of reaction and ManNAc was found inhibitory above 750 mM. The experimental data were then used to evaluate and select different process options, based on design rationale and engineering heuristics. As a result of this approach, process operating windows have been generated, defining possible operating regions for subsequent scale-up. Conventional reactor designs and novel integrated layouts such as in situ product removal were then experimentally assessed. In addition, the option of integration of the epimerisation with the biotransformation (at alkaline pH) has been evaluated. The reactor options were then compared for product, enzyme and downstream separation limiting process scenarios. The methodology of this rational approach to process design was then discussed for the whole class of aqueous phase equilibrium controlled biotransformations. This design approach has advanced in two ways as the result of the findings in this thesis. The characterisation step has been divided further in two sets of experiments, so that unfeasible options were quickly ruled out, accelerating the design procedure. In addition, economic considerations in the form of different processing scenarios have been introduced in the identification of design constraints

Optimal symmetric Tardos traitor tracing schemes

For the Tardos traitor tracing scheme, we show that by combining the symbol-symmetric accusation function of Skoric et al. with the improved analysis of Blayer and Tassa we get further improvements. Our construction gives codes that are up to 4 times shorter than Blayer and Tassa's, and up to 2 times shorter than the codes from Skoric et al. Asymptotically, we achieve the theoretical optimal codelength for Tardos' distribution function and the symmetric score function. For large coalitions, our codelengths are asymptotically about 4.93% of Tardos' original codelengths, which also improves upon results from Nuida et al.Comment: 16 pages, 1 figur

Accelerated process development and stockpile for MERS, LASSA AND NIPAH viral vaccine

CEPI (the Coalition for Epidemic Preparedness Innovation) was launched in January 2017. The global need for CEPI emerged after the devastating Ebola crisis in 2014/15 that caused over 11,000 deaths and had an economic impact of at least $2.8 billion in the worst-affected countries alone[1]. The collective response to Ebola had fallen short, and it was evident we needed a better system to produce proven vaccines against known epidemic threats. A year ago at Davos, the governments of India and Norway and Guinea; the Bill & Melinda Gates Foundation, Wellcome and the World Economic Forum backed the creation of CEPI, an innovative partnership of public, private, philanthropic and civil society organizations, to provide a global insurance policy to defend against future epidemics. Through a call for proposal to vaccine developers, CEPI launched a portfolio of projects covering the development of MERS, Lassa and Nipah viral vaccines, based on WHO blueprint list of pathogens and in addition, a number of platform technologies are currently evaluated for the development of rapid response against unknown pathogens. In this paper, we introduce the CEPI process development, the vaccine technologies and stockpile strategies for MERS, Lassa and Nipah candidates in the CEPI portfolio. The cell lines, process and scale up portfolio strategies will be reviewed for emergency settings vs conventional vaccine process development. The emergency stockpile development strategy will be presented in this paper. In addition, we also highlight the critical areas of dialogue with regulatory authorities for the enhancement of use of experimental vaccine candidates in emergency settings for efficacy trials. [1] http://www.worldbank.org/en/topic/macroeconomics/publication/2014-2015-west-africa-ebola-crisis-impact-updat

Asymptotically false-positive-maximizing attack on non-binary Tardos codes

We use a method recently introduced by Simone and Skoric to study accusation probabilities for non-binary Tardos fingerprinting codes. We generalize the pre-computation steps in this approach to include a broad class of collusion attack strategies. We analytically derive properties of a special attack that asymptotically maximizes false accusation probabilities. We present numerical results on sufficient code lengths for this attack, and explain the abrupt transitions that occur in these results

A adaptação da rádio informativa à pandemia: o caso da Antena 1

Relatório de estágio de mestrado apresentado à Escola Superior de Comunicação Social como parte dos requisitos para obtenção de grau de mestre em Jornalismo.O presente relatório pretende expor as atividades desenvolvidas no âmbito de um estágio realizado na Antena 1 e compreender o modo como esta rádio, perante todos os desafios, se adaptou à situação de pandemia COVID-19. Através da experiência do estágio, da aplicação de inquéritos aos profissionais da rádio e de entrevistas semiestruturadas, tentámos perceber que alterações é que as rotinas de produção jornalísticas sofreram, de que forma é que a tomada de decisão se viu afetada, se a qualidade do conteúdo noticioso teve algum decréscimo e o modo como os jornalistas da Antena 1 percecionaram todas estas mudanças. Este relatório de estágio pretende, além de relatar as experiências vividas durante o seu decorrer, fazer uma análise à adaptação que as exigências da pandemia COVID-19 apresentaram a um meio que sempre mostrou resiliência e se superou em situações de catástrofes e desgraças, como aquela em que ainda hoje vivemos.ABSTRACT: The following report intends to present the activities developed during an internship at Antena 1 as well as to understand how this Portuguese radio has adapted to all the challenges the COVID-19 pandemic has arisen. Through the internship experience, inquiries and semistructured interviews made to radio professionals, we tried to understand what changes occurred in the journalistic production routines, how was the decision-making process affected, if the news content’s quality decreased and how did the journalists from Antena 1 perceive all these changes. This internship report aims not only to describe the activities developed, but also to analyze how this radio has adapted to the demands of the COVID-19 pandemic, knowing that radio itself has always shown resilience and overcome itself in catastrophic situations like the one we still live in.N/

Avaliação do desperdício alimentar numa instituição de suporte a seniores

[Resumo][Abstract

Sliding set-points of immune responses for therapy of autoimmunity

Although recent developments in the treatment of autoimmune disease have dramatically improved patient outcomes, these medications are not curative. Two studies in this issue demonstrate the feasibility of curing spontaneous autoimmunity in animal models via short-term enhancement of naturally arising regulatory T (T reg) cells, a subset of CD4+ T cells needed for maintaining self-tolerance. Importantly, these therapies seemed to generate a new equilibrium, or “set-point,” at which self-tissue damage no longer occurred long after the drug was eliminated from the body

One-pot bio-synthesis: N-acetyl-d-neuraminic acid production by a powerful engineered whole-cell catalyst

Whole cell biocatalysis is an important tool for pharmaceutical intermediates synthesis, although it is hindered by some shortcomings, such as high cost and toxicity of inducer, mass transfer resistance caused by cell membrane and side reactions. Whole-cell catalysis using N-acetyl-d-glucosamine 2-epimerase (EC 5.1.3.8) and N-acetyl-d-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) is a promising approach for the production of Neu5Ac, a potential precursor of many anti-viral drugs. A powerful catalyst was developed by packaging the enzymes in an engineered bacterium and using a safe temperature-induced vector. Since the mass transfer resistance and the side reactions were substantially reduced, a high Neu5Ac amount (191 mM) was achieved. An efficient method was also presented, which allows one-pot synthesis of Neu5Ac with a safe and economic manner. The results highlight the promise of large-scale Neu5Ac synthesis and point at a potential of our approach as a general strategy to improve whole-cell biocatalysis