In vitro reconstitution of the molecular mechanisms of vesicle tethering and membrane fusion

Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes

Gravity and Matter with Asymptotic Safety

Building a consistent Quantum Theory of Gravity is one of the most challenging aspects of modern theoretical physics. In the past couple of years, new attempts have been made along the path of ``asymptotic safety'' through the use of Exact Renormalisation Group Equations, which hinge on the existence of a non-trivial fixed point of the flow equations. We will first summarize the major results that have been obtained along these lines, then we will consider the effect of introducing matter fields into the theory. Our analyses show that in order to preserve the existence of the fixed point one must satisfy some constraints on the matter content of the theory.Comment: 5 pages, 3 figures; talk given at "Renormalization Group and Anomalies in Gravitation and Cosmology", Ouro Preto, Brazil, March 200

Design thinking and acceptance requirements for designing gamified software.

Gamification is increasingly applied to engage people in performing tool-supported collaborative tasks. From previous experiences we learned that available gamification guidelines are not sufficient, and more importantly that motivational and acceptance aspects need to be considered when designing gamified software applications. To understand them, stakeholders need to be involved in the design process. This paper aims to (i) identify key requirements for designing gamified solutions, and (ii) understand if existing methods (partially fitting those requirements) can be selected and combined to provide a comprehensive gamification design method. We discuss a set of key requirements for a suitable gamification design method. We illustrate how to select and combine existing methods to define a design approach that fits those requirements using Design Thinking and the Agon framework. Furthermore, we present a first empirical evaluation of the integrated design method, with participants including both requirements analysts and end-users of the gamified software. Our evaluation offers initial ideas towards a more general, systematic approach for gamification design

Contraints on Matter from Asymptotic Safety

Recent studies of the ultraviolet behaviour of pure gravity suggest that it admits a non-Gaussian attractive fixed point, and therefore that the theory is asymptotically safe. We consider the effect on this fixed point of massless minimally coupled matter fields. The existence of a UV attractive fixed point puts bounds on the type and number of such fields.Comment: 5 pages, 2 figures, revtex4; introduction expande

Emerging influenza strains in the last two decades: A threat of a new pandemic?

In the last 20 years, novel non-seasonal influenza viruses have emerged, most of which have originated from birds. Despite their apparent inability to cause pandemics, with the exception of H1N1 swine influenza virus, these viruses still constitute a constant threat to public health. While general concern has decreased after the peak of the H5N1 virus, in recent years several novel reassorted influenza viruses (e.g., H7N9, H9N2, H10N8) have jumped the host-species barrier and are under surveillance by the scientific community and public health systems. It is still unclear whether these viruses can actually cause pandemics or just isolated episodes. The purpose of this review is to provide an overview of old and novel potential pandemic strains of recent decades

On the Ultraviolet Behaviour of Newton's constant

We clarify a point concerning the ultraviolet behaviour of the Quantum Field Theory of gravity, under the assumption of the existence of an ultraviolet Fixed Point. We explain why Newton's constant should to scale like the inverse of the square of the cutoff, even though it is technically inessential. As a consequence of this behaviour, the existence of an UV Fixed Point would seem to imply that gravity has a built-in UV cutoff when described in Planck units, but not necessarily in other units.Comment: 8 pages; CQG class; minor changes and rearrangement

Asymptotic Safety of Gravity Coupled to Matter

Nonperturbative treatments of the UV limit of pure gravity suggest that it admits a stable fixed point with positive Newton's constant and cosmological constant. We prove that this result is stable under the addition of a scalar field with a generic potential and nonminimal couplings to the scalar curvature. There is a fixed point where the mass and all nonminimal scalar interactions vanish while the gravitational couplings have values which are almost identical to the pure gravity case. We discuss the linearized flow around this fixed point and find that the critical surface is four-dimensional. In the presence of other, arbitrary, massless minimally coupled matter fields, the existence of the fixed point, the sign of the cosmological constant and the dimension of the critical surface depend on the type and number of fields. In particular, for some matter content, there exist polynomial asymptotically free scalar potentials, thus providing a solution to the well-known problem of triviality.Comment: 18 pages,typeset with revtex

New potential biomarkers for early chronic kidney disease diagnosis in patients with different glucose tolerance status

Background: The purpose of the present study was to investigate the role of oxidative stress, platelet activation, and endocan levels in renal dysfunction in normal glucose tolerance (NGT) patients with 1-h plasma glucose values ≥155 mg/dl (NGT ≥ 155), compared to NGT < 155, impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2DM) newly diagnosed subjects. We enlisted 233 patients subjected to an oral glucose tolerance test (OGTT). Materials and methods: The serum levels of platelet activation (glycoprotein VI and sP-selectin), oxidative stress biomarkers (8-isoprostane and Nox-2), and endocan were evaluated using an ELISA test. Results: Among NGT < 155 patients and the T2DM group, there was a statistically significant increase in 8-isoprostane (p < 0.0001), Nox-2 (p < 0.0001), glycoprotein VI (p < 0.0001), and sP-selectin (p < 0.0001) serum levels. Higher serum endocan levels were found with the worsening of metabolic profile (p < 0.0001); specifically, NGT ≥ 155 patients presented higher serum endocan values when compared to NGT < 155 patients (p < 0.0001). From the multivariate linear regression analysis, 1-h glucose resulted in the major predictor of estimated glomerular filtration rate (e-GFR) justifying 23.6% of its variation (p < 0.0001); 8-isoprostane and Nox-2 added respectively another 6.0% (p < 0.0001) and 3.2% (p = 0.001). Conclusion: Our study confirmed the link between 1-h post-load glucose ≥155 mg/dl during OGTT and the possible increased risk for chronic kidney disease (CKD) in newly diagnosed patients. The novelty is that we demonstrated a progressive increase in oxidative stress, platelet activation, and serum endocan levels with the worsening of metabolic profile, which becomes evident early during the progression of CKD