Evolutionary Psychology (EP) tends to be associated with a Massively Modular (MM) cognitive architecture. I argue that EP favors a non-MM cognitive architecture. The main point of dispute is whether central cognition, such as abstract reasoning, exhibits domain-general properties. Partisans of EP argue that domain-specific modules govern central cognition, for it is unclear how the cognitive mind could have evolved domain-generality. In response, I defend a distinction between exogenous and endogenous selection pressures, according to which exogenous pressures tend to select for domain-specificity, whereas the latter, endogenous pressures, select in favor of domain-generality. I draw on models from brain network theory to motivate this distinction, and also to establish that a domain-general, non-MM cognitive architecture is the more parsimonious adaptive solution to endogenous pressures.

Este trabalho propõe um modelo de otimização dinâmico para o controle da infestação por plantas daninhas usando aplicação seletiva de herbicida em um sistema anual de colheita da cultura do milho. A densidade do banco de sementes da população de plantas daninhas a frequência dos alelos dominante e recessivo são tomadas como variáveis de estado. A variável de controle é dada pela função de dose-resposta. O objetivo é reduzir o uso de herbicida, maximizar o lucro em um período pré-determinado de tempo e minimizar os impactos ambientais causados pelo uso excessivo de herbicida. O modelo de otimização leva em consideração a diminuição da eficácia do herbicida ao longo do tempo causada pela pressão seletiva. O problema de otimização dinâmico envolve variáveis inteiras e contínuas que foram modeladas como um problema de programação não linear inteiro misto (MINLP). O problema MINLP foi resolvido por enumeração implícita usando o método branch and bound. Simulações numéricas de uma estratégia ótima ilustra o manejo da planta daninha Bidens subalternans em uma plantação de milho com utilização da seleção de uma sequência de dois herbicidas. A solução obtida é comparada com a solução do problema com aplicação de uma sequência de somente um único herbicida. / A dynamic optimization model for weed infestation control using selective herbicide application in a corn crop system is presented. The seed bank density of the weed population and frequency of dominant and recessive alleles are taken as state variables of the growing cycle. The control variable is taken as the dose response-function. The goal is to reduce herbicide use, maximize profit in a pre-determined period of time and minimize the environmental impacts caused by excessive use of herbicides. The dynamic optimization model takes into account the decreased herbicide efficacy over time due to weed resistance evolution caused by selective pressure. The dynamic optimization problem involves integer and continuous variables which are modeled as a mixed integer nonlinear programming (MINLP) problem. The MINLP problem was solved by an implicit enumeration known as branch and bound method. Numerical simulations for a case study illustrate the management of the Bidens subalternans in a corn crop by selecting a sequence of two herbicides. The solution presented is compared to the solution of a sequence of only one herbicide.

Este trabalho propõe um modelo de otimização dinâmico para o controle da infestação por plantas daninhas usando aplicação seletiva de herbicida em um sistema anual de colheita da cultura do milho. A densidade do banco de sementes da população de plantas daninhas a frequência dos alelos dominante e recessivo são tomadas como variáveis de estado. A variável de controle é dada pela função de dose-resposta. O objetivo é reduzir o uso de herbicida, maximizar o lucro em um período pré-determinado de tempo e minimizar os impactos ambientais causados pelo uso excessivo de herbicida. O modelo de otimização leva em consideração a diminuição da eficácia do herbicida ao longo do tempo causada pela pressão seletiva. O problema de otimização dinâmico envolve variáveis inteiras e contínuas que foram modeladas como um problema de programação não linear inteiro misto (MINLP). O problema MINLP foi resolvido por enumeração implícita usando o método branch and bound. Simulações numéricas de uma estratégia ótima ilustra o manejo da planta daninha Bidens subalternans em uma plantação de milho com utilização da seleção de uma sequência de dois herbicidas. A solução obtida é comparada com a solução do problema com aplicação de uma sequência de somente um único herbicida. / A dynamic optimization model for weed infestation control using selective herbicide application in a corn crop system is presented. The seed bank density of the weed population and frequency of dominant and recessive alleles are taken as state variables of the growing cycle. The control variable is taken as the dose response-function. The goal is to reduce herbicide use, maximize profit in a pre-determined period of time and minimize the environmental impacts caused by excessive use of herbicides. The dynamic optimization model takes into account the decreased herbicide efficacy over time due to weed resistance evolution caused by selective pressure. The dynamic optimization problem involves integer and continuous variables which are modeled as a mixed integer nonlinear programming (MINLP) problem. The MINLP problem was solved by an implicit enumeration known as branch and bound method. Numerical simulations for a case study illustrate the management of the Bidens subalternans in a corn crop by selecting a sequence of two herbicides. The solution presented is compared to the solution of a sequence of only one herbicide.

Submitted by Débora de Oliveira Latorre (deboraolatorre@gmail.com) on 2018-09-18T18:32:45Z No. of bitstreams: 1 Debora de Oliveira Latorre_ Dissertation.pdf: 3050527 bytes, checksum: 150c83efeaa3123bb2b38f3187b2e760 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-09-18T19:29:22Z (GMT) No. of bitstreams: 1 latorre_do_dr_botfca.pdf: 3050527 bytes, checksum: 150c83efeaa3123bb2b38f3187b2e760 (MD5) / Made available in DSpace on 2018-09-18T19:29:22Z (GMT). No. of bitstreams: 1 latorre_do_dr_botfca.pdf: 3050527 bytes, checksum: 150c83efeaa3123bb2b38f3187b2e760 (MD5) Previous issue date: 2017-09-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os herbicidas são um dos fatores mais importantes que vem consideravelmente contribuindo no aumento na proteção das culturas, devido sua inovação no controle de plantas daninhas ao longo dos últimos 70 anos. O uso continuo de um mesmo ingrediente ativo ou modo de ação impõe uma alta pressão de seleção em uma população de plantas daninhas e a seleção de indivíduos resistentes a herbicidas pode ocorrer. A intensidade da seleção imposta pelos herbicidas e a frequência inicial de indivíduos resistentes a herbicidas dentro de uma população de plantas daninhas são fatores chave importantes no processo de evolução da resistência. Fluxo gênico via pólen, sementes e propágulos vegetativos são uma potencial fonte de distribuição de resistência a herbicidas, como previamente reportado em Conyza canadensis e Amaranthus ssp. Conyza canadensis e Amaranthus ssp são potencialmente capazes de transferir genes que conferem resistência a herbicidas via pólen e/ou sementes, por produzirem pólen que pode ser disseminado a longas distancia e grande número de sementes. Os objetivos gerais dos estudos realizados foram caracterizar o nível de resistência de duas espécies de plantas daninhas de Nebraska, Estados Unidos da América. Um primeiro estudo em casa de vegetação foi conduzido para caracterizar o nível de resistência a glyphosate de populações de buva coletadas em áreas não cultivadas foi conduzido. Experimentos de dose-resposta com 9 doses de glyphosate e 28 populações de buva foram avaliados. Um segundo estudo em casa de vegetação foi conduzido para caracterizar o nível de uma população de caruru resistente a 2,4-D a diferentes formulações de herbicidas fenóxicos. De acordo com o primeiro estudo de dose-resposta, menos de sete por cento das populações de Conyza canadensis em áreas de pastagem próximas a áreas de cultivo expressaram “resistência prática” a glyphosate (plantas sobreviventes a dose de glyphosate mais usual em Nebraska – 1,260 g ae ha-1). Baseado em nossos resultados, foi detectado baixa frequência de resistência a glyphosate em populações de Conyza canadensis em áreas de pastagem de Nebraska, indicando que indivíduos resistentes a glyphosate dispersos das áreas de cultivo não são o biótipo predominante nessas áreas. Os resultados do segundo estudo mostraram que a população de Amarantus tuberculatus resistente a 2,4-D foi significativamente mais suscetíveis às formulações dos herbicidas Dicamba DGA, Dicamba DMA, Corasil, 2,4-DP, e 2,4-DP-p, enquanto sobreviveram a altas doses dos herbicidas 2,4-D 2EHE, 2,4-D EE, 2,4-DB, MCPB, MCPA, MCPA 2EHE, CMPP e CMPP-p. / Herbicides are one of the most important factors that have contributed to protect crop yields. This is due to innovative weed control over the last 70 years. The over-reliance on a single herbicide active ingredient or mode of action impose a high selection pressure on a weed population and the selection of herbicide-resistant individual plants may occur. The intensity of selection imposed by herbicides and the initial frequency of herbicide resistant in a weed population play a major role in the herbicide resistance evolution. Gene flow by pollen, seed, and vegetative propagules have the potential to move herbicide-resistant weed species, as reported previous reported in Conyza canadensis and Amaranthus genus. Conyza canadensis and Amaranthus tuberculatus are potentially able to proliferate herbicide resistance by pollen and/or seeds due to be prolific seed producer and its pollen are capable to be disseminated for long distances. The general objectives of these studies were to characterize the herbicide resistance level of two weed species in Nebraska, United States. A greenhouse study was performed to characterize the fold of glyphosate resistance in horseweed populations from non-crop areas. Dose-response experiments with 28 horseweed populations were evaluated across nine glyphosate rates. A second greenhouse study was performed to characterize the level of a 2,4-D-resistant waterhemp population resistance to various auxinic herbicides. According to the first dose-response study, less than seven percent of the rangeland Conyza canadensis populations screened expressed “practical” resistance to glyphosate (plants surviving to most common glyphosate rate used in Nebraska of 1,260g ae ha-1). Therefore, low frequency of GR in horseweed populations was detected in Nebraska rangeland indicating that GR individuals dispersed from row crops into rangeland are not the predominant biotype in these non-row crop areas. For the second study, the results showed that 2,4-D-WR population were significantly more sensitive to Dicamba DGA, Dicamba DMA, Corasil, 2,4-DP, and 2,4-DP-p herbicides formulations, whereas survived to the higher doses of 2,4-D 2EHE, 2,4-D EE, 2,4-DB, MCPB, MCPA, MCPA 2EHE, CMPP and CMPP-p. The founds on this studied showed the 2,4-D-WR population exhibits cross-resistance to 2,4-D 2EHE, 2,4-D EE, 2,4-DB, MCPB, MCPA, MCPA 2EHE, CMPP and CMPP-p herbicides. / 006860/2015-00

Les populations de rongeurs sont responsables de nombreux problèmes économiques et sanitaires. Pour ces raisons certains rongeurs sont qualifiés de nuisibles, tels que le rat brun, le rat noir et la souris domestique. En conséquence, leurs populations doivent être gérées. Cette gestion implique des mesures sanitaires et écologiques strictement indispensables mais passe aussi par l'utilisation de produits chimiques toxiques. Dans le cadre de la lutte chimique, les AVKs sont actuellement les molécules les plus utilisées. Ces AVKs inhibent la coagulation sanguine et sont à l'origine d'hémorragies mortelles. La cible pharmacologique des AVK est l'enzyme VKOR, codée par le gène vkorc1. Cependant, l'utilisation intensive des AVKs depuis les années 1950 a eu pour conséquence, chez les rongeurs, la sélection de résistance à ces molécules. La résistance de cible a été bien étudiée chez le rat brun mais est très peu connue chez la souris domestique et le rat noir / Rodent populations are responsible for many economic and health problems. For these reasons different rodents species are called pests and particularly brown rats, house mice and black rats. Their populations need to be managed. This management involves health and environmental measures strictly necessary but is also based on the use of toxic chemicals. As part of chemical control, the AVKs are currently the most commonly used molecules. These AVKs inhibit blood coagulation and are the cause of fatal bleeding. The pharmacological target of AVK is the VKOR enzymatic activity, encoded by the vkorc1 gene. However, the intensive use of AVKs since the 1950s has led to the development of resistance to these molecules. The target resistance has been well studied in the brown rat, but little is known in the house mouse and the black rat

Les éléments transposables sont des séquences génomiques capables de se répliquer et de se déplacer dans les génomes. Leur capacité à s’insérer près des gènes et à produire des réarrangements chromosomiques par recombinaison entre copies, font des éléments transposables des agents mutagènes. Les éléments transposables sont de plus capables de modifier l’expression des gènes voisins grâce aux régions promotrices qu’ils possèdent. Les éléments transposables ont été trouvés dans la plupart des génomes dans lesquels ils ont été recherchés. Ils forment ainsi 45 % du génome de l’homme et peuvent représenter jusqu’à 90 % du génome de certaines plantes. Dans la première partie de ma thèse, je me suis penché sur les facteurs qui déterminent la distribution de ces éléments. Je me suis intéressé à un facteur particulier, qui est la fonction des gènes dans le voisinage des insertions d’éléments transposables. Dans la deuxième partie, j’ai essayé de déterminer l’impact de l’altération des modifications épigénétiques (modifications d’histones plus précisément) associées aux différents composants géniques, dont les éléments transposables, sur la variation de l’expression des gènes en condition tumorale. / Transposable elements are genomic sequences able to replicate themselves and to move within genomes. Their ability to integrate near genes and to produce chromosomal rearrangements by recombination between copies, make transposable elements mutagens. Moreover, transposable elements are able to alter the expression of neighboring genes through their promoter regions. Transposable elements form 45% of the human genome and may represent up to 90% of certain plant genomes. In the first part of my thesis, I examined the factors that determine the distribution of these elements. I have been interested in a particular factor, which is the function of the genes in the vicinity of transposable element insertions. In the second part, I determined the impact of epigenetic modifications alterations (histone modifications) in different gene components, including transposable elements, on the variation of gene expression in tumoral conditions.

Les troubles du spectre autistique (TSA) touchent approximativement 1% de la population générale. Ces troubles se caractérisent par un déficit de la communication sociale, ainsi que des comportements stéréotypés et des intérêts restreints. Plusieurs gènes impliqués dans le déterminisme des TSA ont été identifiés, comme par exemple les gènes NLGN3-4X, NRXN1-3 et SHANK1-3. Au cours des années précédentes, les TSA ont été considérés comme un ensemble complexe de troubles monogéniques. Cependant, les études récentes du génome complet suggèrent la présence de gènes modificateurs (« multiple hits model »). La dyslexie est caractérisée par un trouble dans l’apprentissage de la lecture et de l’écriture qui touche 5--‐15% de la population générale. Les facteurs génétiques impliqués restent pour l’instant inconnus car seuls des gènes ou loci candidats ont été identifiés. Mon projet de thèse avait pour objectif de poursuivre l’identification des facteurs génétiques impliqués dans les TSA et de découvrir un premier facteur génétique pour la dyslexie. Pour cela, deux types de populations ont été étudiés : d’une part des patients atteints de TSA (N>600) provenant de France, de Suède et des Iles Faroe, d’autre part des patients atteints de dyslexie (N>200) provenant de France, en particulier une famille de 11 personnes atteintes sur 3 générations. J’ai utilisé à la fois la technologie des puces à ADN Illumina (600 K et 5M) et le séquençage complet du génome humain pour effectuer des analyses de liaison et d’association. Pour les TSA, grâce aux analyses de CNVs, j’ai pu identifier des gènes candidats pour l’autisme et confirmer l’association de plusieurs gènes synaptiques avec l’autisme. En particulier, l’étude d’une population de 30 patients des îles Faroe a pu confirmer l’implication des gènes NLGN1 et NRXN1 dans l’autisme et identifier un nouveau gène candidat IQSEC3. En parallèle, j’ai exploréPRRT2 localisé en 16p11.2. PRRT2 code pour un membre du complexe SNARE synaptique qui permet la libération des vésicules synaptiques. Je n’ai pas pu mettre en évidence d’association avec les TSA, mais j’ai montré que ce gène important pour certaines maladies neurologiques était sous pression de sélection différente selon les populations. Pour la dyslexie, j’ai effectué une analyse de liaison (méthode des lod-scores) pour une grande famille de 11 individus atteints sur trois générations. Cette étude a permis d’identifier CNTNAP2 comme un gène de vulnérabilité à la dyslexie. Cette découverte est importante car ce même gène est aussi associé aux TSA. Par contre, aucune des 20 variations rares découvertes par le séquençage complet du génome n’est localisée dans les parties codantes du gène. Plusieurs variations localisées dans des régions régulatrices sont candidates. En conclusion, les résultats de ma thèse ont permis d’identifier des gènes candidats pour les TSA, de confirmer le rôle des gènes synaptiques dans ce trouble, de montrer pour la première fois grâce à une analyse de liaison le rôle de CNTNAP2 dans la dyslexie. / Autism spectrum disorders (ASD) affect 1% of the general population. These disorders are characterized by deficits in social communication as well as stereotyped behaviors and restricted interests. Several genes involved in the determination of ASD have been identified, such as NLGN3-4, NRXN1-3 and SHANK1-3. In the previous years, ASD have been considered as a complex set of monogenic disorders. Recent studies on the complete genome nevertheless suggest the presence of modifier genes ("multiple hits model"). Dyslexia is characterized by difficulties in learning to read and write. It affects 5-15 % of the general population. Genetic factors involved remain unknown. Only candidate genes or loci have been identified. My thesis had two main objectives: pursuing the identification of genetic factors involved in ASD, and discovering a first genetic factor for dyslexia. I therefore studied two types of populations: on the one hand a group of patients with ASD (N > 600) from France, Sweden and the Faroe Islands, and on the other hand another group of patients with dyslexia (N > 200) from France, and more specifically a family of 11 people followed over 3 generations. I used both Illumina microarrays technology (600K and 5M) and the complete human genome sequencing to conduct linkage and association analyses. Regarding ASD, CNVs (copy number variants) analyses allowed me to confirm the association of several synaptic genes with autism and to identify new candidate genes. In particular, the study of a population of 30 patients from the Faroe Islands confirmed the involvement of NLGN1 and NRXN1 genes in autism and identified a new candidate gene, IQSEC3. At the same time, I explored PRRT2 located in 16p11.2. PRRT2 encodes a member of the synaptic SNARE complex that allows the release of synaptic vesicles. I have not been able to demonstrate any association with ASD, but I showed that this gene, which is important for some neurological diseases, was under different selection pressures according to the population considered. Regarding dyslexia, I realized a linkage analysis (lod-score method) for a large family of 11 individuals, with three generations affected. This study identified the CNTNAP2 gene as a vulnerability factor for dyslexia. This finding is important because this gene is also associated with ASD. Nevertheless, none of the 20 rare variations discovered by whole genome sequencing is localized in the coding parts of the gene. Only several variations localized in regulatory regions are robust candidates. To conclude, my findings enabled the identification of new candidate genes for ASD, the confirmation of the role of synaptic genes in this disorder, and the highlight for the first time of the role of CNTNAP2 in dyslexia through linkage analysis.

L’hypothèse de gènes chevauchants codés par le brin antisens des rétrovirus est un concept ancien. Cependant, celui-ci n’a été réellement démontré qu’il y a une dizaine d’années avec la découverte de la protéine HBZ du virus HTLV-1 et les résultats récents sur la protéine ASP du VIH-1. Les nouvelles recherches sur cette protéine ont démontré son expression in vivo, mais sa fonctionnalité est toujours inconnue. Nous avons réalisé ici, à partir d’un jeu de données de plus de 20 000 séquences, les premières analyses bioinformatiques sur l’évolution de ce gène chevauchant. Nous avons alors montré que le gène asp est conservé uniquement dans les séquences du groupe M correspondant au groupe pandémique du VIH-1. Nous démontrons de plus, une corrélation entre la présence de l’ORF ASP et la prévalence des différents groupes et sous-types. Nos analyses phylogénétiques montrent que l’apparition de l’ORF ASP est concomitante avec l’émergence de la pandémie du groupe M. Du fait du chevauchement de gènes, l’analyse de la pression de sélection induite par la protéine ASP a impliqué l’utilisation de modèles et de méthodes d’analyses spécifiques. Situé sur la phase -2, ce chevauchement entraîne une correspondance des troisièmes bases des codons de chaque gène. Si on considère un gène « fixe », les contraintes mécaniques induites par ce gène sont très importantes et le gène chevauchant dispose de très peu de flexibilité. Il est alors important d’identifier la pression de sélection propre au gène situé sur la phase -2, et montrer ainsi que sa présence et sa conservation ne sont pas seulement dues aux contraintes induites par le gène fixe. Pour cela, nous avons tout d’abord montré par des analyses de simulations de séquences, à l’aide d’un modèle à codon, que la présence de l’ORF ASP n’est pas due au hasard. Nous avons ensuite développé une méthode d’analyse évolutive basée sur l’étude des mutations silencieuses pour le gène fixe (ici le gène env) entraînant l’apparition ou la disparition des codons Start et Stop sur le gène chevauchant. L’application de cette méthode au gène asp montre qu’il existe bien une pression de sélection induite par la protéine ASP. / The hypothesis of overlapping genes encoded by the antisense strand of the retrovirus is an old concept. However, this one has been really demonstrated that with the discovery of the HBZ protein of HTLV-1 virus, a dozen of years ago and the recent results on the ASP protein of HIV-1. New research on this protein has demonstrated its expression in vivo, but its functionality is still unknown. We performed here, from a data set of more than 20,000 sequences, the first bioinformatic analyses on the evolution of this overlapping gene. We showed that the asp gene is conserved only in the M group sequences corresponding to the pandemic group of HIV-1. Moreover, we demonstrated a correlation between the presence of the ASP ORF and the prevalence of the various groups and subtypes. Our phylogenetic analyses showed that the appearance of the ASP ORF is concomitant with the emergence of the pandemic M group. Because of the overlapping of the genes, the analysis of the selection pressure induced by the ASP protein involved the use of models and specific analysis methods. Located in the frame -2, this overlap induces a correspondence of the third base codon of each gene. If we consider a "fixed" gene, the mechanical constraints induced by this "fixed" gene are very important and the overlapping gene has very little flexibility. Then, it is important to identify the selection pressure of the gene which is in the frame -2 and show that its presence and conservation are not only due to mechanical constraints induced by the "fixed" gene. For this, we first demonstrated by sequence simulation analysis, using a codon model, that the presence of the ASP ORF is not due to chance. Then, we developed a method of evolutionary analysis based on the study of synonymous mutations in the "fixed" gene (here the env gene) causes the appearance or disappearance of start and stop codons in the overlapping gene. When we applied this method to the asp gene, it showed that there is a selection pressure induced by the ASP protein.

Little is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages.

Establishing group structure in complex networks is potentially very useful since nodes belonging to the same module can often be related by commonalities in their biological function. However, module detection in complex networks poses a challenging problem and has sparked a great deal of interest in various disciplines in recent years [5]. In real networks, which can be quite complex, we have no idea about the true number of modules that exist. Furthermore, the structure of the modules may be hierarchical meaning they may be further divided into sub-modules and so forth. Many attempts have been made to deal with these problems and because the involved methods vary considerably they have been difficult to compare [5]. The objectives of this thesis are (i) to create and implement a new algorithm that will identify modules in complex networks and reconstruct the network in such a way so as to maximize modularity, (ii) to evaluate the performance of a new method, and compare it to a popular method based on a simulated annealing algorithm, and (iii) to apply the new method, and a comparator method, to analyze the metabolic network of the bacterial genus Listeria, an important pathogen in both agricultural and human clinical settings.