Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.

Aphid resistance in brazilian hexaploid wheat does not correlate with levels of the benzoxazinoid natural product DIMBOA.

2016

Influence of the natural environment on health: evaluation of different environmental contexts (urban, green spaces) and intervention proposals

Urbanization has grown during the last decades, with an increase in population concentrated in cities. Cities are usually relatively nature-poor, and the loss of green urban space likely leads to less contact with the natural world for urban dwellers. It is known that the natural environment could provide important advantages, and the loss of contact with this type of environment has potential negative impacts on the quality of life. The use of green urban space demonstrated stronger benefits for mental health and stress reduction. In general, exposure to green urban space is linked to a reduction in mortality rates, due to the promotion of a healthy lifestyle. Green urban space could be an optimal environment in which to perform physical activity. Undertaking regular physical activity is one of the major determinants of health. The benefits of exercise have been widely demonstrated through a wide range of studies. Benefits are linked to the treatment and prevention of most chronic and non-communicable diseases, that are not contagious, but they are usually long-lasting. Regular physical activity could reduce mental health problems, such as anxiety. The World Health Organization proposed to improve physical activity programs through the implementation of interventions in green urban spaces. Green urban space provides a safe, accessible, and attractive place to perform physical activity. All the interventions aimed to promote the practice of physical activity and to reduce sedentary behavior are important. It is well known that physical activity has several positive effects, a great amount of the population remains inactive. A good strategy could be to show people how integrated physical activity into their all-day life, for example through the use of green urban space or active commuting. The results in the present thesis showed the effectiveness of performing physical activity in a natural environment and of active commuting.

Toward wellbeing: investigation of traditional, classical and innovative approaches to improve population and environmental health

As the word population continues to grow and global resources are limited, the WHO definition of health is difficult to achieve for a large part of the population. Humanity is facing the need to improve both environmental and human wellbeing. This can be done through careful planning and management of natural resources, ensuring food safety and reducing and converting wastes. This work aims to contribute to the improvement of population and environmental health exploring different research fields: urban park ecosystem services, food chemical risk assessment and agri-food by-product valorization. To highlight the importance of urban parks and their ecosystem services, an ethnobotanical study was carried out in the Ausa urban park in Rimini, using a citizen science approach. The results showed that Ausa Park is an important focal point for plant gatherers in Rimini, as it allows for plant foraging and contributes to preserve the knowledge of the use of plants. Two food safety studies were carried out, looking at the exposure of Poles to bisphenol A through the consumption of soft drinks and to cadmium through the consumption of chocolate bars. The results, compared with EFSA’s scientific opinion, show that the exposure of the Polish population to BPA is of health concern, while cadmium is not. In the agri-food by-product valorization, a green extraction method was optimized to recover valuable phenolic compounds from red-fleshed apple pomace; moreover, the possibility of recovering pectin from the residue was evaluated. Furthermore, valuable compounds in four different types of wheat milling by-products, considered as an alternative source of bioactive compounds with potential human health benefits, were investigated. In conclusion, this work produced usable data in urban green area management and planning, in food chemical risk assessment and in business production decisions, thus contributing to improving environmental and people wellbeing.

Intestinal Anti-Inflammatory Activity of Baccharis dracunculifolia in the Trinitrobenzenesulphonic Acid Model of Rat Colitis

Baccharis dracunculifolia DC (Asteraceae) is a Brazilian medicinal plant popularly used for its antiulcer and anti-inflammatory properties. This plant is the main botanical source of Brazilian green propolis, a natural product incorporated into food and beverages to improve health. The present study aimed to investigate the chemical profile and intestinal anti-inflammatory activity of B. dracunculifolia extract on experimental ulcerative colitis induced by trinitrobenzenosulfonic acid (TNBS). Colonic damage was evaluated macroscopically and biochemically through its evaluation of glutathione content and its myeloperoxidase (MPO) and alkaline phosphatase activities. Additional in vitro experiments were performed in order to test the antioxidant activity by inhibition of induced lipid peroxidation in the rat brain membrane. Phytochemical analysis was performed by HPLC using authentic standards. The administration of plant extract (5 and 50 mgkg(-1)) significantly attenuated the colonic damage induced by TNBS as evidenced both macroscopically and biochemically. This beneficial effect can be associated with an improvement in the colonic oxidative status, since plant extract prevented glutathione depletion, inhibited lipid peroxidation and reduced MPO activity. Caffeic acid, p-coumaric acid, aromadendrin-4-O-methyl ether, 3-prenyl-p-coumaric acid, 3,5-diprenyl-p-coumaric acid and baccharin were detected in the plant extract.

Skewed Distribution of Circulating Activated Natural Killer T (NKT) Cells in Patients with Common Variable Immunodeficiency Disorders (CVID)

Common variable immunodeficiency disorder (CVID) is the commonest cause of primary antibody failure in adults and children, and characterized clinically by recurrent bacterial infections and autoimmune manifestations. Several innate immune defects have been described in CVID, but no study has yet investigated the frequency, phenotype or function of the key regulatory cell population, natural killer T (NKT) cells. We measured the frequencies and subsets of NKT cells in patients with CVID and compared these to healthy controls. Our results show a skewing of NKT cell subsets, with CD4+ NKT cells at higher frequencies, and CD8+ NKT cells at lower frequencies. However, these cells were highly activated and expression CD161. The NKT cells had a higher expression of CCR5 and concomitantly expression of CCR5+CD69+CXCR6 suggesting a compensation of the remaining population of NKT cells for rapid effector action.

Genome-wide SNP genotyping highlights the role of natural selection in Plasmodium falciparum population divergence

Background: The malaria parasite Plasmodium falciparum exhibits abundant genetic diversity, and this diversity is key to its success as a pathogen. Previous efforts to study genetic diversity in P. falciparum have begun to elucidate the demographic history of the species, as well as patterns of population structure and patterns of linkage disequilibrium within its genome. Such studies will be greatly enhanced by new genomic tools and recent large-scale efforts to map genomic variation. To that end, we have developed a high throughput single nucleotide polymorphism (SNP) genotyping platform for P. falciparum. Results: Using an Affymetrix 3,000 SNP assay array, we found roughly half the assays (1,638) yielded high quality, 100% accurate genotyping calls for both major and minor SNP alleles. Genotype data from 76 global isolates confirm significant genetic differentiation among continental populations and varying levels of SNP diversity and linkage disequilibrium according to geographic location and local epidemiological factors. We further discovered that nonsynonymous and silent (synonymous or noncoding) SNPs differ with respect to within-population diversity, interpopulation differentiation, and the degree to which allele frequencies are correlated between populations. Conclusions: The distinct population profile of nonsynonymous variants indicates that natural selection has a significant influence on genomic diversity in P. falciparum, and that many of these changes may reflect functional variants deserving of follow-up study. Our analysis demonstrates the potential for new high-throughput genotyping technologies to enhance studies of population structure, natural selection, and ultimately enable genome-wide association studies in P. falciparum to find genes underlying key phenotypic traits.

Genetic variability and natural selection at the ligand domain of the Duffy binding protein in brazilian Plasmodium vivax populations

Background: Plasmodium vivax malaria is a major public health challenge in Latin America, Asia and Oceania, with 130-435 million clinical cases per year worldwide. Invasion of host blood cells by P. vivax mainly depends on a type I membrane protein called Duffy binding protein (PvDBP). The erythrocyte-binding motif of PvDBP is a 170 amino-acid stretch located in its cysteine-rich region II (PvDBP(II)), which is the most variable segment of the protein. Methods: To test whether diversifying natural selection has shaped the nucleotide diversity of PvDBP(II) in Brazilian populations, this region was sequenced in 122 isolates from six different geographic areas. A Bayesian method was applied to test for the action of natural selection under a population genetic model that incorporates recombination. The analysis was integrated with a structural model of PvDBP(II), and T-and B-cell epitopes were localized on the 3-D structure. Results: The results suggest that: (i) recombination plays an important role in determining the haplotype structure of PvDBP(II), and (ii) PvDBP(II) appears to contain neutrally evolving codons as well as codons evolving under natural selection. Diversifying selection preferentially acts on sites identified as epitopes, particularly on amino acid residues 417, 419, and 424, which show strong linkage disequilibrium. Conclusions: This study shows that some polymorphisms of PvDBP(II) are present near the erythrocyte-binding domain and might serve to elude antibodies that inhibit cell invasion. Therefore, these polymorphisms should be taken into account when designing vaccines aimed at eliciting antibodies to inhibit erythrocyte invasion.

Cloud point extraction and concentration of carbaryl from natural waters

An improved procedure is proposed for determination of the pesticide carbaryl in natural waters based on double cloud point extraction. The clean up step was carried out only with Triton X-114 in alkaline medium in order to avoid the use of toxic organic solvents as well as to minimise waste generation. Cloud point preconcentration of the product of the reaction of the analyte with p-aminophenol and cetyltrimethylammonium bromide was explored to increase sensitivity and improve the detection limit. Linear response was achieved within 10 and 500 mu g L-1 and the apparent molar absorptivity was estimated as 4.6 x 105 L mol-1 cm-1. The detection limit was estimated as 7 mu g L-1 at the 99.7% confidence level and the coefficient of variation was 3.4% (n = 8). Recoveries within 91 and 99% were estimated for carbaryl spiked water samples. The results obtained for natural water samples were in agreement with those achieved by the batch of spectrophotometric procedure at the 95% confidence level. The proposed procedure is then a simple, fast, inexpensive and greener alternative for carbaryl determination.

Fermentation of cacao (Theobroma cacao L.) seeds with a hybrid Kluyveromyces marxianus strain improved product quality attributes

Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage (`sweatings`) from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

NATURAL LIGHT IN PINEAPPLE (Ananas comosus L) MICROPROPAGATION

Shoot tips of Ananas comosus `Imperial were rooted in vitro under two environments (artificial and natural light) and after two months the plantlets were transferred to commercial substrate (Plantmax (R)) in a greenhouse. Plant growth and leaf anatomy were evaluated at 0, 7, 15, 30 and 60-days during acclimatization. The in vitro rooting under natural light provides better agronomic and anatomical performances of Ananas comosus plants, with the benefit of saving electric energy for artificial lumination in vegetal tissue culture laboratories.

Enrollment in Physical Education Is Associated With Health-Related Behavior Among High School Students

METHODS: A total of 4210 students attending public high schools in Pernambuco (northeast of Brazil) were selected using random 2-stage cluster sampling. Data were collected by using the Global School-based Student Health Survey. The independent variable was the frequency of participation in PE classes, whereas physical activity, television viewing, smoking, and alcohol, fruit, vegetables and soda consumption were dependent variables. Logistic regressions were carried out to perform crude and adjusted analysis of the association between enrollment in PE classes and health-related behaviors. RESULTS: Sixty-five percent of students do not take part in PE classes, with a significantly higher proportion among females (67.8%). It was observed that enrollment in PE classes was positively associated with physical activity, TV viewing, and fruit consumption, but was negatively associated with soda drinking. The likelihood of reporting being active and eating fruit on a daily basis was 27% and 45% higher, respectively, among those who participate in at least 2 classes per week in comparison with those who do not. Students who participate in PE classes had 28-30% higher likelihood of reporting lower TV viewing during week days. CONCLUSIONS: Findings suggest that higher levels of enrollment in PE classes could play a role in the promotion of health-related behaviors among high school students.

The sorption of toxic elements onto natural zeolite, synthetic goethite and modified powdered block carbon

Inorganic elements analyses of Carapicuiba lake reveal that As, Cr, Pb and Mn are above the recommended drinking water standards. The mean total concentrations of toxic elements in surface water decrease in the order Mn > Cr > Pb > As. At elevated concentrations, toxic elements like Cr can accumulate in soils and enter the food chain, leading to serious health hazards and threatening the long-term sustainability of the local ecosystem. Absorbing materials has often been used to improve water quality. In this investigation three types of material were studied: the natural zeolite (mordenite); synthetic goethite and the powdered block carbon modified. The adsorption of Pb(2+) and Mn(2+) onto natural zeolite as a function of their concentrations was studied at 24 degrees C by varying the metal concentration from 100 to 400 mg L(-1) while keeping all other parameters constant. The low-cost zeolites removed Pb from water without any pretreatment at pH values <6. The maximum adsorption attained was as follows: Pb(2+) 78.7% and Mn(2+) 19.6%. The modified powdered block carbon effectively removed As(V) and Cr(VI) while goethite removed more chromate than arsenate in the pH range 5-6. Results of this study will be used to evaluate the application these materials for the treatment of the Carapicuiba lake`s water.

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection

Inheritance of resistance to Puccinia psidii G. Winter in a eucalyptus interspecific hybrid progeny evaluated under conditions of natural infection Rust caused by the fungus Puccinia psidii is currently the most important disease of eucalyptus. It is widely disseminated in Brazil, and causes serious damage in nurseries and plantation areas. The identification of resistant germplasm along with knowledge of the genetic basis of resistance heredity are the first requirements for the success of breeding programs aiming to develop resistant varieties. Earlier studies carried out under controlled conditions suggested a monogenic control as well as the participation of at least two genes promoting resistance to rust. The goal of this study was to evaluate the resistance to P. psidii under field conditions in fourteen progenies from controlled crosses and self-crosses among four hybrid clones of Eucalyptus grandis Hill ex Maiden x Eucalyptus urophylla ST Blake that contrast for resistance to the fungus. Results indicated that resistance could be explained by one locus with main effects and at least three different alleles. However, loci with minor effects may influence the resistance, since variation on severity classes was observed. Differences in segregation of resistance between reciprocal crosses were not observed, indicating absence of cytoplasmic effects.

Fauna of plant-parasitic nematodes in natural and cultivated areas of the Amazon forest, Mato Grosso State, Brazil

The aim of this work was to study the diversity of the fauna of plant-parasitic nematodes in preserved areas of the Amazon forest, Mato Grosso state (MT), and to assess the effect of agricultural land use on plant-parasitic nematode communities. Soil and root samples were collected in each location in the late spring during the rainy season of 2005, in two areas of primary vegetation in Nova Maringa (Northwest) and Guaranta do Norte (North) and two adjacent areas planted with teak trees (Tectona grandis) and pasture (Brachiaria brizantha). Four-teen taxa of plant-parasitic nematodes were identified at species level: Discocriconemella degrissei, D. limitanea, Dolichodorus minor, Helicotylenchus erythrinae, H. pseudorobustus, Meloidogyne exigua, M javanica, Mesocriconema ornata, Paratrichodorus minor, Pratylenchus loosi, P zeae, Rotylenchus caudaphasmidius, Xiphinema ensiculiferum and X luci (for the first report of this in Brazil) and five at genus level (Atalodera sp., Hemicriconemoides sp., Meloidogyne sp., Paratylenchus sp., and Trophotylenchulus sp). These taxa, mainly those from primary vegetation, belong to families with different parasitic behavior, probably due to great plant diversity in the Amazon forest. Comparison between the two preserved areas revealed low index of similarity, as a consequence of the endemic flora in the Amazon forest, and no similarity was observed between preserved native vegetation and adjacent cultivated areas, demonstrating the high influence of agricultural activity on the plant-parasitic nematode communities. There is evidence of recent introduction of plant-parasitic nematodes in these cultivated areas; therefore measures should be taken to prevent the loss of economic sustainability in Amazonian soils. Keywords: abundance, Amazonia, diversity, Brachiaria brizantha, plant-parasitic nematode fauna, Tectona grandis.