Filling in gaps of Drosophila melanogaster urate degradation metabolic pathway using metabolomics approaches: towards the core metabolome of the fruit fly

The primary goal of systems biology is to integrate complex omics data, and data obtained from traditional experimental studies in order to provide a holistic understanding of organismal function. One way of achieving this aim is to generate genome-scale metabolic models (GEMs), which contain information on all metabolites, enzyme-coding genes, and biochemical reactions in a biological system. Drosophila melanogaster GEM has not been reconstructed to date. Constraint-free genome-wide metabolic model of the fruit fly has been reconstructed in our lab, identifying gaps, where no enzyme was identified and metabolites were either only produced or consume. The main focus of the work presented in this thesis was to develop a pipeline for efficient gap filling using metabolomics approaches combined with standard reverse genetics methods, using 5-hydroxyisourate hydrolase (5-HIUH) as an example. 5-HIUH plays a role in urate degradation pathway. Inability to degrade urate can lead to inborn errors of metabolism (IEMs) in humans, including hyperuricemia. Based on sequence analysis Drosophila CG30016 gene was hypothesised to encode 5- HIUH. CG30016 knockout flies were examined to identify Malpighian tubules phenotype, and shortened lifespan might reflect kidney disorders in hyperuricemia in humans. Moreover, LC-MS analysis of mutant tubules revealed that CG30016 is involved in purine metabolism, and specifically urate degradation pathway. However, the exact role of the gene has not been identified, and the complete method for gap filling has not been developed. Nevertheless, thanks to the work presented here, we are a step closer towards the development of a gap-filling pipeline in Drosophila melanogaster GEM. Importantly, the areas that require further optimisation were identified and are the focus of future research. Moreover, LC-MS analysis confirmed that tubules rather than the whole fly were more suitable for metabolomics analysis of purine metabolism. Previously, Dow/Davies lab has generated the most complete tissue-specific transcriptomic atlas for Drosophila – FlyAtlas.org, which provides data on gene expression across multiple tissues of adult fly and larva. FlyAtlas revealed that transcripts of many genes are enriched in specific Drosophila tissues, and that it is possible to deduce the functions of individual tissues within the fly. Based on FlyAtlas data, it has become clear that the fly (like other metazoan species) must be considered as a set of tissues, each 2 with its own distinct transcriptional and functional profile. Moreover, it revealed that for about 30% of the genome, reverse genetic methods (i.e. mutation in an unknown gene followed by observation of phenotype) are only useful if specific tissues are investigated. Based on the FlyAtlas findings, we aimed to build a primary tissue-specific metabolome of the fruit fly, in order to establish whether different Drosophila tissues have different metabolomes and if they correspond to tissue-specific transcriptome of the fruit fly (FlyAtlas.org). Different fly tissues have been dissected and their metabolome elucidated using LC-MS. The results confirmed that tissue metabolomes differ significantly from each other and from the whole fly, and that some of these differences can be correlated to the tissue function. The results illustrate the need to study individual tissues as well as the whole organism. It is clear that some metabolites that play an important role in a given tissue might not be detected in the whole fly sample because their abundance is much lower in comparison to other metabolites present in all tissues, which prevent the detection of the tissue-specific compound.

MULTI-BAND BOSE HUBBARD MODELS AND EFFECTIVE THREE-BODY INTERACTIONS

Experiments with ultracold atoms in optical lattice have become a versatile testing ground to study diverse quantum many-body Hamiltonians. A single-band Bose-Hubbard (BH) Hamiltonian was first proposed to describe these systems in 1998 and its associated quantum phase-transition was subsequently observed in 2002. Over the years, there has been a rapid progress in experimental realizations of more complex lattice geometries, leading to more exotic BH Hamiltonians with contributions from excited bands, and modified tunneling and interaction energies. There has also been interesting theoretical insights and experimental studies on “un- conventional” Bose-Einstein condensates in optical lattices and predictions of rich orbital physics in higher bands. In this thesis, I present our results on several multi- band BH models and emergent quantum phenomena. In particular, I study optical lattices with two local minima per unit cell and show that the low energy states of a multi-band BH Hamiltonian with only pairwise interactions is equivalent to an effec- tive single-band Hamiltonian with strong three-body interactions. I also propose a second method to create three-body interactions in ultracold gases of bosonic atoms in a optical lattice. In this case, this is achieved by a careful cancellation of two contributions in the pair-wise interaction between the atoms, one proportional to the zero-energy scattering length and a second proportional to the effective range. I subsequently study the physics of Bose-Einstein condensation in the second band of a double-well 2D lattice and show that the collision aided decay rate of the con- densate to the ground band is smaller than the tunneling rate between neighboring unit cells. Finally, I propose a numerical method using the discrete variable repre- sentation for constructing real-valued Wannier functions localized in a unit cell for optical lattices. The developed numerical method is general and can be applied to a wide array of optical lattice geometries in one, two or three dimensions.

Scedo-Select III: a new semi-selective culture medium for detection of the Scedosporium apiospermum species complex

International audience

Development and pharmacological evaluation of ropivacaine-2-hydroxypropyl-beta-cyclodextrin inclusion complex

Ropivacaine (RVC) is an enantiomerically pure local anesthetic (LA) largely used in surgical procedures, which presents physico-chemical and therapeutic properties similar to those of bupivacaine (BPV), but associated to less systemic toxicity This study focuses on the development and pharmacological evaluation of a RVC in 2-hydroxypropyl-beta-cyclodextrin (HP-P-CD) inclusion complex. Phase-solubility diagrams allowed the determination of the association constant between RVC and HP-beta-CD (9.46 M-1) and showed an increase on RVC solubility upon complexation. Release kinetics revealed a decrease on RVC release rate and reduced hemolytic effects after complexation. (onset at 3.7 mM and 11.2 mM for RVC and RVCHP-beta-CD, respectively) were observed. Differential scanning calorimetry (DSC), scanning electron microscopy (SEM) and X-ray analysis (X-ray) showed the formation and the morphology of the complex. Nuclear magnetic resonance (NMR) and job-plot experiments afforded data regarding inclusion complex stoichiometry (1:1) and topology. Sciatic nerve blockade studies showed that RVCHP-beta-CD was able to reduce the latency without increasing the duration of motor blockade, but prolonging the duration and intensity of the sensory blockade (p < 0.001) induced by the LA in mice. These results identify the RVCHP-beta-CD complex as an effective novel approach to enhance the pharmacological effects of RVC, presenting it as a promising new anesthetic formulation. (c) 2007 Elsevier B.V All rights reserved.

On the critical point structure of eigenfunctions belonging to the first nonzero eigenvalue of a genus two closed hyperbolic surface

We develop a method based on spectral graph theory to approximate the eigenvalues and eigenfunctions of the Laplace-Beltrami operator of a compact riemannian manifold -- The method is applied to a closed hyperbolic surface of genus two -- The results obtained agree with the ones obtained by other authors by different methods, and they serve as experimental evidence supporting the conjectured fact that the generic eigenfunctions belonging to the first nonzero eigenvalue of a closed hyperbolic surface of arbitrary genus are Morse functions having the least possible total number of critical points among all Morse functions admitted by such manifolds

Transport in the blood of an anti-tumor water-soluble rutheniun cyclopentadienyl complex: a fluorescence study on albumin binding

Dissertação de mestrado, Qualidade em Análises, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

E-polynomial of SL(2,C)-character varieties of complex curves of genus 3.

We compute the E-polynomials of the moduli spaces of representations of the fundamental group of a complex curve of genus g = 3 into SL(2, C), and also of the moduli space of twisted representations. The case of genus g = 1, 2 has already been done in [12]. We follow the geometric technique introduced in [12], based on stratifying the space of representations, and on the analysis of the behaviour of the E-polynomial under fibrations.

Análisis de las variables de marketing que afectan al valor del cliente. La permanencia como variable controlable

To know how marketing variables affect customer value is essential for a company in order to be market and customer oriented, and to improve investment efficiency in both attracting and retaining customers. Thus, the assessment of the influence of marketing variables in customer value is of prime importance. This is recognized in many empirical studies of these variables, which address the impact of a single variable (or sets of a few variables) on customer value. A comprehensive, integrated assessment of all marketing variables and their interdependencies is an arduous and complex task for researchers and marketing managers. This research proposes a theoretical model of customer value that takes into account all significant marketing variables that have been partially addressed in empirical investigations of other researchers. These marketing variables include brand and reputation, point of sale, employees, price, termination fee commitment, discounts, complementarity of products, experiences, emotions, perceived value, quality, satisfaction, switching costs, and loyalty. The model incorporates the relationship between each variable with retention and with customer value as well as the relationships between them. A special focus is placed on the empirical analysis of the termination fee commitment and its relationship with customer value. This variable is widely used in the telecommunication’s industry for its influence on customer retention from the moment of purchase. However, there is strikingly little research in this topic. A large customer database of a telecommunications company containing five years information about 63.165 customers is used for this purpose. Multivariate linear regression and ANOVA method are applied...

Rapamycin reverses age-related increases in mitochondrial ROS production at complex I, oxidative stress, accumulation of mtDNA fragments inside nuclear DNA, and lipofuscin level, and increases autophagy, in the liver of middle-aged mice

Rapamycin consistently increases longevity in mice although the mechanism of action of this drug is unknown. In the present investigation we studied the effect of rapamycin on mitochondrial oxidative stress at the same dose that is known to increase longevity in mice (14 mg of rapamycin/kg of diet). Middle aged mice (16 months old) showed significant age-related increases in mitochondrial ROS production at complex I, accumulation of mtDNA fragments inside nuclear DNA, mitochondrial protein lipoxidation, and lipofuscin accumulation compared to young animals (4 months old) in the liver. After 7 weeks of dietary treatment all those increases were totally or partially (lipofuscin) abolished by rapamycin, middle aged rapamycin-treated animals showing similar levels in those parameters to young animals. The decrease in mitochondrial ROS production was due to qualitative instead of quantitative changes in complex I. The decrease in mitochondrial protein lipoxidation was not due to decreases in the amount of highly oxidizable unsaturated fatty acids. Rapamycin also decreased the amount of RAPTOR (of mTOR complex) and increased the amounts of the PGC1-α and ATG13 proteins. The results are consistent with the possibility that rapamycin increases longevity in mice at least in part by lowering mitochondrial ROS production and increasing autophagy, decreasing the derived final forms of damage accumulated with age which are responsible for increased longevity. The decrease in lipofuscin accumulation induced by rapamycin adds to previous information suggesting that the increase in longevity induced by this drug can be due to a decrease in the rate of aging. © 2016 Elsevier Inc.

Temperature-averaged and total free-free Gaunt factors for κ and Maxwellian distributions of electrons

Aims. Optically thin plasmas may deviate from thermal equilibrium and thus, electrons (and ions) are no longer described by the Maxwellian distribution. Instead they can be described by κ-distributions. The free-free spectrum and radiative losses depend on the temperature-averaged (over the electrons distribution) and total Gaunt factors, respectively. Thus, there is a need to calculate and make available these factors to be used by any software that deals with plasma emission. Methods. We recalculated the free-free Gaunt factor for a wide range of energies and frequencies using hypergeometric functions of complex arguments and the Clenshaw recurrence formula technique combined with approximations whenever the difference between the initial and final electron energies is smaller than 10−10 in units of z2Ry. We used double and quadruple precisions. The temperature- averaged and total Gaunt factors calculations make use of the Gauss-Laguerre integration with 128 nodes. Results. The temperature-averaged and total Gaunt factors depend on the κ parameter, which shows increasing deviations (with respect to the results obtained with the use of the Maxwellian distribution) with decreasing κ. Tables of these Gaunt factors are provided.

Employment of Real-Time/FPGA Architectures for Test and Control of Automotive Engines

Nowadays the production of increasingly complex and electrified vehicles requires the implementation of new control and monitoring systems. This reason, together with the tendency of moving rapidly from the test bench to the vehicle, leads to a landscape that requires the development of embedded hardware and software to face the application effectively and efficiently. The development of application-based software on real-time/FPGA hardware could be a good answer for these challenges: FPGA grants parallel low-level and high-speed calculation/timing, while the Real-Time processor can handle high-level calculation layers, logging and communication functions with determinism. Thanks to the software flexibility and small dimensions, these architectures can find a perfect collocation as engine RCP (Rapid Control Prototyping) units and as smart data logger/analyser, both for test bench and on vehicle application. Efforts have been done for building a base architecture with common functionalities capable of easily hosting application-specific control code. Several case studies originating in this scenario will be shown; dedicated solutions for protype applications have been developed exploiting a real-time/FPGA architecture as ECU (Engine Control Unit) and custom RCP functionalities, such as water injection and testing hydraulic brake control.

Nickel dependent carcinogenesis and infections: structural and biophysical characterization of NDRG1 and SgSrnR

In prokaryotic organisms, lower eukaryotes and plants, some important biological reactions are catalyzed by nickel-dependent enzymes, making this metal ion essential microelement for their life. On the other hand, excessive concentration of nickel into the cell, or prolonged exposure to nickel compounds, has toxic effects in living organisms. In addition, nickel has been classified by IARC as Group I human carcinogen, because of the correlation between its inhalation and increased incidence of nasal and lung cancers. The aim of this work was to investigate the nickel impact on human health, considering both its direct role on human cells and its indirect effect as essential element for human important bacteria. In humans, nickel induces N-myc downstream regulated gene 1 (NDRG1) expression, recently proposed as new target in cancer therapy. CD, light scattering and ITC were applied on the recombinant full-length protein and its C-terminal intrinsically disordered domain, for studying the NDRG1 structural and functional properties. In particular, the fold and dynamics of the C-terminal region were examined by NMR spectroscopy and site-directed spin labeling coupled to EPR, showing the features of an intrinsically disordered region. In nickel-dependent bacteria, nickel metabolism is strictly regulated, through the activity of different transcription factors. In Streptomyces griseus the expression of two superoxide dismutases (SODs) is antagonistically regulated by nickel thanks to the transcriptional complex SgSrnR/SgSrnQ. The SgSrnR protein was heterologously expressed and its activity as possible nickel sensor studied. DNaseI footprinting and β-galactosidase gene reporter assays revealed that SgSrnR functions as transcriptional activator, prompting the hypothesis of a new model to describe the activity of this complex. In addition, ITC, NMR and X-ray crystallography demonstrated that SgSrnR presents the fold typical of ArsR/SmtB transcription factors and low metal binding affinity, non compatible with a role as a nickel-sensor, function probably played by its partner SgSrnQ.

Molecular modelling of organic functional materials

The investigation of the mechanisms lying behind the (photo-)chemical processes is fundamental to address and improve the design of new organic functional materials. In many cases, dynamics simulations represent the only tool to capture the system properties emerging from complex interactions between many molecules. Despite the outstanding progresses in calculation power, the only way to carry out such computational studies is to introduce several approximations with respect to a fully quantum mechanical (QM) description. This thesis presents an approach that combines QM calculations with a classical Molecular Dynamics (MD) approach by means of accurate QM-derived force fields. It is based on a careful selection of the most relevant molecular degrees of freedom, whose potential energy surface is calculated at QM level and reproduced by the analytic functions of the force field, as well as by an accurate tuning of the approximations introduced in the model of the process to be simulated. This is made possible by some tools developed purposely, that allow to obtain and test the FF parameters through comparison with the QM frequencies and normal modes. These tools were applied in the modelling of three processes: the npi* photoisomerisation of azobenzene, where the FF description was extended to the excited state too and the non-adiabatic events were treated stochastically with Tully fewest switching algorithm; the charge separation in donors-acceptors bulk heterojunction organic solar cells, where a tight-binding Hamiltonian was carefully parametrised and solved by means of a code, also written specifically; the effect of the protonation state on the photoisomerisation quantum yield of the aryl-azoimidazolium unit of the axle molecule of a rotaxane molecular shuttle. In each case, the QM-based MD models that were specifically developed gave noteworthy information about the investigated phenomena, proving to be a fundamental key for a deeper comprehension of several experimental evidences.

Mathematical methods to analyze and interpret calcium signals of astrocytes

Astrocytes are the most numerous glial cell type in the mammalian brain and permeate the entire CNS interacting with neurons, vasculature, and other glial cells. Astrocytes display intracellular calcium signals that encode information about local synaptic function, distributed network activity, and high-level cognitive functions. Several studies have investigated the calcium dynamics of astrocytes in sensory areas and have shown that these cells can encode sensory stimuli. Nevertheless, only recently the neuro-scientific community has focused its attention on the role and functions of astrocytes in associative areas such as the hippocampus. In our first study, we used the information theory formalism to show that astrocytes in the CA1 area of the hippocampus recorded with 2-photon fluorescence microscopy during spatial navigation encode spatial information that is complementary and synergistic to information encoded by nearby "place cell" neurons. In our second study, we investigated various computational aspects of applying the information theory formalism to astrocytic calcium data. For this reason, we generated realistic simulations of calcium signals in astrocytes to determine optimal hyperparameters and procedures of information measures and applied them to real astrocytic calcium imaging data. Calcium signals of astrocytes are characterized by complex spatiotemporal dynamics occurring in subcellular parcels of the astrocytic domain which makes studying these cells in 2-photon calcium imaging recordings difficult. However, current analytical tools which identify the astrocytic subcellular regions are time consuming and extensively rely on user-defined parameters. Here, we present Rapid Astrocytic calcium Spatio-Temporal Analysis (RASTA), a novel machine learning algorithm for spatiotemporal semantic segmentation of 2-photon calcium imaging recordings of astrocytes which operates without human intervention. We found that RASTA provided fast and accurate identification of astrocytic cell somata, processes, and cellular domains, extracting calcium signals from identified regions of interest across individual cells and populations of hundreds of astrocytes recorded in awake mice.

Models for the assessment of resilience in water distribution networks

The following thesis work focuses on the use and implementation of advanced models for measuring the resilience of water distribution networks. In particular, the functions implemented in GRA Tool, a software developed by the University of Exeter (UK), and the functions of the Toolkit of Epanet 2.2 were investigated. The study of the resilience and failure, obtained through GRA Tool and the development of the methodology based on the combined use of EPANET 2.2 and MATLAB software, was tested in a first phase, on a small-sized literature water distribution network, so that the variability of the results could be perceived more clearly and with greater immediacy, and then, on a more complex network, that of Modena. In the specific, it has been decided to go to recreate a mode of failure deferred in time, one proposed by the software GRA Tool, that is failure to the pipes, to make a comparison between the two methodologies. The analysis of hydraulic efficiency was conducted using a synthetic and global network performance index, i.e., Resilience index, introduced by Todini in the years 2000-2016. In fact, this index, being one of the parameters with which to evaluate the overall state of "hydraulic well-being" of a network, has the advantage of being able to act as a criterion for selecting any improvements to be made on the network itself. Furthermore, during these analyzes, was shown the analytical development undergone over time by the formula of the Resilience Index. The final intent of this thesis work was to understand by what means to improve the resilience of the system in question, as the introduction of the scenario linked to the rupture of the pipelines was designed to be able to identify the most problematic branches, i.e., those that in the event of a failure it would entail greater damage to the network, including lowering the Resilience Index.