Municipal solid waste management system: decision support through systems analysis

Thesis submitted to the Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia for the degree of Doctor of Philosophy in Environmental Engineering

Using gold nanoparticles in protein crystallography: studies in crystal growth and derivatization

Dissertation for the Master Degree in Structural and Functional Biochemistry

Spatial point pattern analysis of gorilla nest sites in the Kagwene Sanctuary, Cameroon: Towards understanding the nesting behavior of a critically endangered subspecies

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Analysis of the Dynamics and Origin of Epileptic Activity in Patients with Tuberous Sclerosis Evaluated for Surgery of Epilepsy

Objective: The epilepsies associated with the tuberous sclerosis complex (TSC) are very often refractory to medical therapy. Surgery for epilepsy is an effective alternative when the critical link between the localization of seizure onset in the scalp and a particular cortical tuber can be established. In this study we perform analysis of ictal and interictal EEG to improve such link. Methods: The ictal and interictal recordings of four patients with TSC undergoing surgery for epilepsy were submitted to independent component analysis (ICA), followed by source analysis, using the sLORETA algorithm. The localizations obtained for the ictal EEG and for the average interictal spikes were compared. Results: The ICA of ictal EEG produced consistent results in different events, and there was good agreement with the tubers that were successfully removed in three of the four patients (one patient refused surgery). In some patients there was a large discrepancy between the localization of ictal and interictal sources. The interictal activity produced more widespread source localizations. Conclusions: The use of ICA of ictal EEG followed by the use of source analysis methods in four cases of epilepsy and TSC was able to localize the epileptic generators very near the lesions successfully removed in surgery for epilepsy. Significance: The ICA of ictal EEG events may be a useful add-on to the tools used to establish the connection between epileptic scalp activity and the cortical tubers originating it, in patients with TSC considered for surgery of epilepsy.

Visions on high-speed trains: a methodological analysis

Based on the report for the unit “Foresight Analysis Methods” of the PhD program on Technology Assessment in 2013. This unit was supervised by Prof. António Moniz. The paper had meanwhile contributions from the supervisor and Dr. Douglas Robinson.

Algorithms and computational tools for metabolic flux analysis

PhD thesis in Biomedical Engineering

Compositional analysis of biofilms formed by Staphylococcus aureus isolated from food sources

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2016.00390

Quantifying consistency of Event Related Potentials across subjects based on single-trial topographic analysis

Introduction: Non-invasive brain imaging techniques often contrast experimental conditions across a cohort of participants, obfuscating distinctions in individual performance and brain mechanisms that are better characterised by the inter-trial variability. To overcome such limitations, we developed topographic analysis methods for single-trial EEG data [1]. So far this was typically based on time-frequency analysis of single-electrode data or single independent components. The method's efficacy is demonstrated for event-related responses to environmental sounds, hitherto studied at an average event-related potential (ERP) level. Methods: Nine healthy subjects participated to the experiment. Auditory meaningful sounds of common objects were used for a target detection task [2]. On each block, subjects were asked to discriminate target sounds, which were living or man-made auditory objects. Continuous 64-channel EEG was acquired during the task. Two datasets were considered for each subject including single-trial of the two conditions, living and man-made. The analysis comprised two steps. In the first part, a mixture of Gaussians analysis [3] provided representative topographies for each subject. In the second step, conditional probabilities for each Gaussian provided statistical inference on the structure of these topographies across trials, time, and experimental conditions. Similar analysis was conducted at group-level. Results: Results show that the occurrence of each map is structured in time and consistent across trials both at the single-subject and at group level. Conducting separate analyses of ERPs at single-subject and group levels, we could quantify the consistency of identified topographies and their time course of activation within and across participants as well as experimental conditions. A general agreement was found with previous analysis at average ERP level. Conclusions: This novel approach to single-trial analysis promises to have impact on several domains. In clinical research, it gives the possibility to statistically evaluate single-subject data, an essential tool for analysing patients with specific deficits and impairments and their deviation from normative standards. In cognitive neuroscience, it provides a novel tool for understanding behaviour and brain activity interdependencies at both single-subject and at group levels. In basic neurophysiology, it provides a new representation of ERPs and promises to cast light on the mechanisms of its generation and inter-individual variability.

Evidence for transcript networks composed of chimeric RNAs in human cells.

The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network.

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

Dibenzofuran degradation by Sphingomonas wittichii RW1 under environmental stresses

Sphingomonas wittichii is a gram-negative Alpha-proteobacterium, capable of degrading xenobiotic compounds such as dibenzofuran (DBF), dibenzo-p-dioxin, carbazole, 2-hydroxybiphenyl or nitro diphenyl ether herbicides. The metabolism of strain RW1 has been the subject of previous studies and a number of genes involved in DBF degradation have been characterized. It is known that RW1 posseses a unique initial DBF dioxygenase (encoded by the dxnAl gene) that catalyzes the first step in the degradation pathway. None of the organisms known to be able to degrade DBF have a similar dioxygenase, the closest match being the DBF dioxygenase from Rhodococcus sp. with an overall amino acid similarity of 45%. Genes participating in the conversion of the metabolite salicylate via the ortho-cleavage pathway to TCA cycle intermediates were identified as well. Apart from this scarce information, however, there is a lack of global knowledge on the genes that are involved in DBF degradation by strain RW1 and the influence of environmental stresses on DBF-dependent global gene expression. A global analysis is necessary, because it may help to better understand the behaviour of the strain under field conditions and suggest improvements for the current bioaugmentation practice. Chapter 2 describes the results of whole-genome analysis to characterize the genes involved in DBF degradation by RW1. Micro-array analysis allowed us to detect differences in gene transcription when strain RW1 was exposed to DBF. This was complemented by ultra-high throughput sequencing of mutants no longer capable of growing on salicylate and DBF. Some of the genes of the ortho-cleavage pathway were induced 2 to 4 times in the presence of DBF, as well as the initial DBF dioxygenase. However two gene clusters, named 4925 and 5102 were induced up to 19 times in response to DBF induction. The cluster 4925 is putatively participating in a meta-cleavage pathway while the cluster 5102 might be part of a gentisate pathway. The three pathways, ortho-cleavage, meta-cleavage and gentisate pathway seem to be active in parallel when strain RW1 is exposed to DBF, presenting evidence for a redundancy of genes for DBF degradation in the genome of RW1. Chapter 3 focuses on exploiting genetic tools to construct bioreporters representative for DBF degradation in RW1. A set of basic tools for genetic manipulation in Sphingomonas wittichii RW1 was tested and optimized. Both plasmids and mini-transposons were evaluated for their ability to be maintained in RW1 with or without antibiotic selection pressure, and for their ability to lead to fluorescent protein expression in strain RW1 from a constitutive promoter. Putative promoter regions of three of the previously found DBF-induced genes (Swit_4925, Swit_5102 and Swit_4897-dxnAl) were then used to construct eg/^-bioreporters in RW1. Chapter 4 describes the use of the constructed RW1-based bioreporter strains for examining the expression of the DBF degradation pathway genes under microcosm conditions. The bioreporter strains were first exposed to different carbon sources in liquid culture to calibrate the egfp induction. Contrary to our expectations from micro-array analysis only the construct with the promoter from gene cluster 4925 responded to DBF, whereas the other two constructs did not show specific induction with DBF. The response from the bioreporters was subsequently tested for sensitivity to water stress, given that this could have an important impact in soils. Exposure to liquid cultures with decreasing water potential, achieved by NaCl or PEG addition to the growth media, showed that eGFP expression in RW1 from the promoter regions 4925 and 5102 was not directly influenced by water stress, but only through an overall reduction in growth rate. In contrast, expression of eGFP from the dxnAl or an uspA promoter was also directly dependent on the extent of water stress. The RW1 with the 4925 construct was subsequently used in soil microcosms to evaluate DBF bioavailability to the cells in presence or absence of native microbiota or other contaminated material. We found that RW1 could grow on DBF added to soil, but bioreporter expression suggested that competition with native microbiota for DBF intermediates may limit its ability to proliferate to a maximum. Chapter 5 describes the results from the experiments carried out to more specifically detect genes of RW1 that might be implicated in water stress resistance. Hereto we created transposon mutagenesis libraries in RW1, either with a classical mini-Tn5 or with a variant that would express egfp when the transposon would insert in a gene induced under water stress. Classical mutant libraries were screened by replica plating under high and low water stress conditions (achieved by adding NaCl to the agar medium). In addition, we screened for smaller microcolonies formed by mutants in agarose beads that could be analized with flow cytometry. A number of mutants impaired to grow on NaCl-supplemented media were recovered and the transposon insertion sites sequenced. In a second procedure we screened by flow cytometry for mutants with a higher eGFP production after exposure to growth medium with higher NaCl concentrations. Mutants from both libraries rarely overlapped. Discovered gene functions of the transposon insertions pointed to compatible solute synthesis (glutamate and proline), cell membrane synthesis and modification of cell membrane composition. The results obtained in the present study give us a more complete picture of the mechanisms of DBF degradation by S. wittichii RW1, how it reacts to different DBF availability and how the DBF catabolic activity may be affected by the conditions found in contaminated environments. - Sphingomonas wittichii est une alpha-protéobactérie gram-négative, capable de dégrader des composés xénobiotiques tels que le dibenzofurane (DBF), la dibenzo-p-dioxine, le carbazole, le 2-hydroxybiphényle ou les herbicides dérivés du nitro-diphényléther. Le métabolisme de la souche RW1 a fait l'objet d'études antérieures et un certain nombre de gènes impliqués dans la dégradation du DBF ont été caractérisés. Il est connu que RW1 possède une unique dioxygénase DBF initiale (codée par le gène dxnAl) qui catalyse la première étape de la voie de dégradation. Aucun des organismes connus pour être capables de dégrader le DBF n'a de dioxygénase similaire. L'enzyme la plus proche étant la DBF dioxygénase de Rhodococcus sp. avec 45% d'acides aminés conservés. Les gènes qui participent à la transformation du salicylate en métabolites intermédiaires du cycle de Krebs par la voie ort/io-cleavage ont aussi été identifiés. Outre ces informations lacunaires, il y a un manque de connaissances sur l'ensemble des gènes impliqués dans la dégradation du DBF par la souche RW1 ainsi que l'effet des stress environnementaux sur l'expression génétique globale, en présence du DBF. Une analyse globale est nécessaire, car elle peut aider à mieux comprendre le comportement de la souche dans les conditions de terrain et de proposer des améliorations pour l'utilisation de la bio-augmentation comme technique de bio-remédiation. Le chapitre 2 décrit les résultats de l'analyse du génome pour caractériser les gènes impliqués dans la dégradation du DBF par RW1. Une analyse de micro-arrays nous a permis de détecter des différences dans la transcription des gènes lorsque la souche RW1 a été exposée au DBF. L'analyse a été complétée par le criblage à ultra-haut débit de mutants qui n'étaient plus capables de croître avec le salicylate ou le DBF comme seule source de carbone. Certains des gènes de la voie ortho-cleavage, dont la DBF dioxygénase initiale, ont xî été induits 2 à 4 fois, en présence du DBF. Cependant, deux groupes de gènes, nommés 4925 et 5102 ont été induits jusqu'à 19 fois en réponse au DBF. Le cluster 4925 participe probablement dans une voie de meta-cleavage tandis que le cluster 5102 pourrait faire partie d'une voie du gentisate. Les trois voies, ortho-cleavage, meta-cleavage et la voie du gentisate semblent être activées en parallèle lorsque la souche RW1 est exposée au DBF, ce qui représente une redondance de voies pour la dégradation du DBF dans le génome de RW1. Le chapitre 3 se concentre sur l'exploitation des outils génétiques pour la construction de biorapporteurs de la dégradation du DBF par RW1. Un ensemble d'outils de base pour la manipulation génétique dans Sphingomonas wittichii RW1 a été testé et optimisé. Deux plasmides et mini-transposons ont été évalués pour leur capacité à être maintenu dans RW1 avec ou sans pression de sélection par des antibiotiques, et pour leur capacité à exprimer la protéine fluorescente verte (eGFP) dans la souche RW1. Les trois promoteurs des gènes Swit_4925, Swit_5102 et Swit_4897 (dxnAl), induits en réponse au DBF, ont ensuite été utilisés pour construire des biorapporteurs dans RW1. Le chapitre 4 décrit l'utilisation des souches biorapportrices construites pour l'analyse de l'expression des gènes de la voie de dégradation du DBF dans des microcosmes avec différents types de sols. Les souches biorapportrices ont d'abord été exposées à différentes sources de carbone en cultures liquides afin de calibrer l'induction de la eGFP. La construction avec le promoteur du gène 4925 a permis une réponse au DBF. Mais contrairement à nos attentes, basées sur les résultats de l'analyse des micro-arrays, les deux autres constructions n'ont pas montré d'induction spécifique au DBF. La réponse des biorapporteurs a ensuite été testée pour la sensibilité au stress hydrique, étant donné que cela pourrait avoir un impact important dans les microcosmes. La diminution du potentiel hydrique en culture liquide est obtenue par addition de NaCl ou de PEG au milieu de croissance. Nous avons montré que l'expression de la eGFP contrôlée par les promoteurs 4925 et 5102 n'était pas directement influencée par le stress hydrique, mais seulement par une réduction globale des taux de croissance. En revanche, l'expression de la eGFP dépendante des promoteurs dxnAl et uspA était aussi directement dépendante de l'ampleur du stress hydrique. La souche avec la construction 4925 a été utilisée par la suite dans des microcosmes avec différents types de sols pour évaluer la biodisponibilité du DBF en présence ou absence des microbes indigènes et d'autres composés contaminants. Nous avons constaté que RW1 pouvait se développer si le DBF a été ajouté au sol, mais l'expression de la eGFP par le biorapporteur suggère que la compétition avec la microbiota indigène pour les métabolites intermédiaires du DBF peut limiter sa capacité à proliférer de manière optimale. Le chapitre 5 décrit les résultats des expériences réalisées afin de détecter spécifiquement les gènes de RW1 qui pourraient être impliquées dans la résistance au stress hydrique. Ici on a crée des bibliothèques de mutants de RW1 par transposon, soit avec un mini-Tn5 classique ou avec une variante qui exprime la eGFP lorsque le transposon s'insère dans un gène induit par le stress hydrique. Les bibliothèques de mutants ont été criblées par la méthode classique de repiquage sur boîtes, dans des conditions de stress hydrique élevé (obtenu par l'addition de NaCl dans les boîtes). En outre, nous avons criblé des micro¬colonies dans des billes d'agarose qui ont pu être analysées par cytométrie de flux. Un certain nombre de mutants déficients à croître sur des milieux supplémentés avec du NaCl ont été isolés et les sites d'insertion du transposon séquencés. Dans une deuxième procédure nous avons criblé par cytométrie de flux des mutants avec une production de eGFP supérieure, après exposition à un milieu de croissance avec une concentration élevée de NaCl. Les mutants obtenus dans les deux bibliothèques n'étaient pas similaires. Les fonctions des gènes où se trouvent les insertions de transposons sont impliqués dans la synthèse de solutés compatibles (glutamate et de la proline), dans la synthèse de la membrane cellulaire et dans la modification de la composition de la membrane cellulaire. Les résultats obtenus dans la présente étude nous donnent une image plus complète des mécanismes de dégradation du DBF par S. wittichii RW1, comment cette souche réagit à la disponibilité du DBF et comment l'activité catabolique peut être affectée par les conditions rencontrées dans des environnements contaminés.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Dynamic simulation of regulatory networks using SQUAD.

BACKGROUND: The ambition of most molecular biologists is the understanding of the intricate network of molecular interactions that control biological systems. As scientists uncover the components and the connectivity of these networks, it becomes possible to study their dynamical behavior as a whole and discover what is the specific role of each of their components. Since the behavior of a network is by no means intuitive, it becomes necessary to use computational models to understand its behavior and to be able to make predictions about it. Unfortunately, most current computational models describe small networks due to the scarcity of kinetic data available. To overcome this problem, we previously published a methodology to convert a signaling network into a dynamical system, even in the total absence of kinetic information. In this paper we present a software implementation of such methodology. RESULTS: We developed SQUAD, a software for the dynamic simulation of signaling networks using the standardized qualitative dynamical systems approach. SQUAD converts the network into a discrete dynamical system, and it uses a binary decision diagram algorithm to identify all the steady states of the system. Then, the software creates a continuous dynamical system and localizes its steady states which are located near the steady states of the discrete system. The software permits to make simulations on the continuous system, allowing for the modification of several parameters. Importantly, SQUAD includes a framework for perturbing networks in a manner similar to what is performed in experimental laboratory protocols, for example by activating receptors or knocking out molecular components. Using this software we have been able to successfully reproduce the behavior of the regulatory network implicated in T-helper cell differentiation. CONCLUSION: The simulation of regulatory networks aims at predicting the behavior of a whole system when subject to stimuli, such as drugs, or determine the role of specific components within the network. The predictions can then be used to interpret and/or drive laboratory experiments. SQUAD provides a user-friendly graphical interface, accessible to both computational and experimental biologists for the fast qualitative simulation of large regulatory networks for which kinetic data is not necessarily available.

MAMOT: hidden Markov modeling tool.

Hidden Markov models (HMMs) are probabilistic models that are well adapted to many tasks in bioinformatics, for example, for predicting the occurrence of specific motifs in biological sequences. MAMOT is a command-line program for Unix-like operating systems, including MacOS X, that we developed to allow scientists to apply HMMs more easily in their research. One can define the architecture and initial parameters of the model in a text file and then use MAMOT for parameter optimization on example data, decoding (like predicting motif occurrence in sequences) and the production of stochastic sequences generated according to the probabilistic model. Two examples for which models are provided are coiled-coil domains in protein sequences and protein binding sites in DNA. A wealth of useful features include the use of pseudocounts, state tying and fixing of selected parameters in learning, and the inclusion of prior probabilities in decoding. AVAILABILITY: MAMOT is implemented in C++, and is distributed under the GNU General Public Licence (GPL). The software, documentation, and example model files can be found at http://bcf.isb-sib.ch/mamot

Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.

The development of targeted treatment strategies adapted to individual patients requires identification of the different tumor classes according to their biology and prognosis. We focus here on the molecular aspects underlying these differences, in terms of sets of genes that control pathogenesis of the different subtypes of astrocytic glioma. By performing cDNA-array analysis of 53 patient biopsies, comprising low-grade astrocytoma, secondary glioblastoma (respective recurrent high-grade tumors), and newly diagnosed primary glioblastoma, we demonstrate that human gliomas can be differentiated according to their gene expression. We found that low-grade astrocytoma have the most specific and similar expression profiles, whereas primary glioblastoma exhibit much larger variation between tumors. Secondary glioblastoma display features of both other groups. We identified several sets of genes with relatively highly correlated expression within groups that: (a). can be associated with specific biological functions; and (b). effectively differentiate tumor class. One prominent gene cluster discriminating primary versus nonprimary glioblastoma comprises mostly genes involved in angiogenesis, including VEGF fms-related tyrosine kinase 1 but also IGFBP2, that has not yet been directly linked to angiogenesis. In situ hybridization demonstrating coexpression of IGFBP2 and VEGF in pseudopalisading cells surrounding tumor necrosis provided further evidence for a possible involvement of IGFBP2 in angiogenesis. The separating groups of genes were found by the unsupervised coupled two-way clustering method, and their classification power was validated by a supervised construction of a nearly perfect glioma classifier.