Minimal metabolic pathways for ATP and generation of a proton motive force in synthetic cells

In an effort to mimic bacterial life and to build a bacterial cell from the bottom up, we have developed metabolic routes for synthetic cells. These enzymatic pathways could eventually provide a synthetic cell with the energy required to sustain itself and ultimately proliferate. This thesis two of these pathways, as well as methods to monitor them with sensors and external methods. The first pathway provides the synthetic cell with electrical energy. A proton motive force is generated by a pathway consisting of two proteins. A membrane transporter imports malate, which is converted into lactate inside the vesicles. This process increases the pH on the inside of the vesicles, and additionally accumulates a negative charge. The membrane potential that is generated is great enough to potentially import other substrates into the cell or to drive the generation of ATP through the F1F0-ATPsynthetase.The second pathway presented provides the synthetic cell with the universal energy carrier ATP through four proteins in total. The membrane protein imports arginine and exports the waste product ornithine. The three internal proteins convert arginine to citrulline and subsequently ornithine, which phosphatizes ADP in the process, yielding ATP. We show that the generated ATP can be used by another transporter to transport glycine betaine into the vesicles.To monitor the presented pathways, methods are described to monitor the ATP to ADP ratio, pH, membrane potential, production of lactate and the concentrations of arginine, citrulline, ornithine and ammonia

A multinational survey of companion animal veterinary clinicians: How can antimicrobial stewardship guidelines be optimised for the target stakeholder?

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Reward sensitivity in ADHD:what do we know and how can we use it?

Children and adults with attention-deficit/hyperactivity disorder (ADHD) experience concentration problems and/or excessive impulsivity and restlessness. Worldwide, there has already been a lot of research conducted on the role of sensitivity to reward in ADHD. Therefore, this thesis includes systematic literature reviews to summarize knowledge about several aspects of reward sensitivity in ADHD. In the first literature review, we found evidence that children and adolescents with ADHD take more risks on gambling tasks than peers without ADHD, in which they often prefer a less likely, large reward over a more likely, smaller reward. This evidence is weaker for adults with ADHD. A second literature review revealed that children with ADHD often respond in the same way to social rewards than other children and are sometimes even hyperresponsive to social rewards. Thus, children with ADHD may have more difficulty estimating risks but do not necessarily differ in their sensitivity to social rewards. The third literature review concerned a meta-analysis based on 40 years of educational research, assessing the effectiveness of classroom interventions for ADHD. This review showed that classroom interventions using rewards (linked to desired behavior) result in large improvements in behavior of students with symptoms of ADHD. Finally, a survey study among teachers provided important insights into Dutch teachers’ experiences with such evidence-based effective classroom interventions for ADHD. These findings emphasize the need for (the development of) adequate teacher support and for bridging the gap between science and practice

Fluorescence-based sensing of the bioenergetic and physicochemical status of the cell

Fluorescence-based sensors play a fundamental role in biological research. These sensors can be based on fluorescent proteins, fluorescent probes or they can be hybrid systems. The availability of a very large dataset of fluorescent molecules, both genetically encoded and synthetically produced, together with the structural insights on many sensing domains, allowed to rationally design a high variety of sensors, capable of monitoring both molecular and global changes in living cells or in in vitro systems. The advancements in the fluorescence-imaging field helped researchers to obtain a deeper understanding of how and where specific changes occur in a cell or in vitro by combining the readout of the fluorescent sensors with the spatial information provided by fluorescent microscopy techniques. In this review we give an overview of the state of the art in the field of fluorescent biosensors and fluorescence imaging techniques, and eventually guide the reader through the choice of the best combination of fluorescent tools and techniques to answer specific biological questions. We particularly focus on sensors for probing the bioenergetics and physicochemical status of the cell.</p

Provisioning urbanism: a comparative urban-rural zooarchaeology of ancient Southwest Asia

Historically, urban centres are seen as consumers that draw in labour and resources from their rural hinterlands. Zooarchaeological studies of key urban sites in Southwest Asia demonstrate the movement of livestock, but the region-wide application of these findings has not been tested and the logistics of urban provisioning remain poorly understood. Here, the authors analyse zooarchaeological data from 245 sites in the Levant and Mesopotamia to examine patterns of livestock production and consumption over a 5000-year period. They find that although preferences varied over time and space, urban sites consistently relied on rural satellites to overcome local limitations to support their large and diverse populations

Between the Danube and the Deep Blue Sea : zooarchaeological meta-analysis reveals variability in the spread and development of Neolithic farming across the western Balkans

The first spread of farming practices into Europe in the Neolithic period involves two distinct 'streams', respectively around the Mediterranean littoral and along the Danube corridor to central Europe. In this paper we explore variation in Neolithic animal use practices within and between these streams, focusing on the first region in which they are clearly distinct (and yet still in close proximity): the western Balkans. We employ rigorous and reproducible meta-analysis of all available zooarchaeological data from the region to test hypotheses (a) that each stream featured a coherent 'package' of herding and hunting practices in the earliest Neolithic, and (b) that these subsequently diverged in response to local conditions and changing cultural preferences. The results partially uphold these hypotheses, while underlining that Neolithisation was a complex and varied process. A coherent, stable, caprine-based 'package' is seen in the coastal stream, albeit with some diversification linked to expansion northwards and inland. Accounting for a severe, systematic bias in bone recovery methodology between streams, we show that sheep and goats also played a major role across the continental stream in the earliest Neolithic (c.6100-5800 BC). This was followed by a geographically staggered transition over c.500 years to an economy focused on cattle, with significant levels of hunting in some areas – a pattern we interpret in terms of gradual adaptation to local conditions, perhaps mediated by varying degrees of cultural conservatism. Subsequent westward expansion carried with it elements of this new pattern, which persisted through the middle and late Neolithic