Дисертації з теми "Microbial behavior"

Thesis (Ph.D)--Civil and Environmental Engineering, Georgia Institute of Technology, 2008.
Committee Chair: Santamarina, J. Carlos; Committee Member: Burns, Susan; Committee Member: Frost, David; Committee Member: Mitchell, James; Committee Member: Rix, Glenn; Committee Member: Sobecky, Patricia.

Microbial life in nature is typically associated with two distinctive features: (i) the formation of densely packed groups, mostly on surfaces, within which social interactions are rife, and (ii) mixing with multiple strains and species, which brings with it the potential for strong evolutionary conflict. However, most microbiology still rests upon the study of single genetic backgrounds, often in shaking flasks. There is a need then to better integrate the natural ecology and evolution of microbes into microbiological studies. My thesis combines ecoevolutionary theory with the empirical study of both population dynamics and single-cell behavior to understand social interactions within microbial communities. This systems biology approach suggests that competition, not cooperation, is central to understanding diverse microbial communities and can explain key social phenotypes including cross-feeding, biofilm formation and antibiotic resistance. Specifically, I study the theoretical limits for cooperation within microbial communities and conclude that natural selection will rarely favor cooperation between genotypes; I show that antibiotic competition between strains is an important driver of biofilm formation, which is consistent with the idea of competition sensing (the ability to use antibiotics, or more generally any form of biotic stress, as a cue to detect and respond to ecological competition); I establish the behavioral and genetic basis of chemotaxis on surfaces at the single-cell level and discuss how this phenotype can be useful in the social milieu of microbial communities where positioning is a major determinant for evolutionary competition; and finally, I show that bacteria are able to climb antibiotic gradients via twitching chemotaxis and achieve an unprecedented level of resistance in a few hours by simply modulating their physiology. Again, and as for antibiotic-induced biofilm formation, I argue that this perplexing phenotype has its natural roots in competition sensing. The study of microbial interactions has emphasized the importance of cooperation between cells of a single genotype. My thesis shows the need to consider the other side of microbial interactions: evolutionary competition. A cell will commonly face intense competition from other strains and species, and only by appreciating this evolutionary conflict will we fully understand microbes and their complex communities.

A novel method for the bioremediation of phenanthrene using the fungus Penicillium frequentans was utilised to remove phenanthrene (200 mg kg\(^{-1}\)) from soil containing both metals and phenanthrene, over 29 days. Bioremediation of phenanthrene and its effects on trace metal behaviour has been investigated. Metal behaviour studied includes metal speciation and the kinetics of exchange between solution and solid phase and plant uptake of the more labile and mobile, and potentially more bioavailable metal species. Phenanthrene removal by P. frequentans was optimised in terms of both soil water and nutrient composition. Slightly lower removal rates were obtained using P. frequentans alone (73%) and plants alone (67%). However, the highest phenanthrene removal (77%) was obtained using both fungus and plant. Assessment of the metal behaviour before and after phenanthrene biodegradation showed that the removal of phenanthrene by either fungal or mixed fungal and native microflora resulted in an increased flux of metal from solid to solution, an increased pool of potentially bioavailable and toxic metal species and increased plant uptake to both Echinochloa polystachia and Triticum aestivum, by factors of 4-13. In the presence of plants alone, metal mobilisation and uptake increased by smaller factors. In some cases, there was no increase in metal mobilisation and a maximal increase of 2 was found in Ni and Pb. These results highlight the impact of bioremediation process on metal behaviour. In addition, it is suggested that phytoremediation and not bioaugmentation using P. frequentans is the best overall option to obtain a considerable phenanthrene removal, reducing the increased pool of potentially bioavailable and toxic metal species

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2014.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 83-90).
This thesis presents design, manufacturing, testing, and modeling of a laminar-flow microbial fuel cell. Novel means were developed to use graphite and other bulk-scale materials in a microscale device without loosing any properties of the bulk material. Micro-milling techniques were optimized for use on acrylic to achieve surface roughness averages as low as Ra = 100nm for a 55 [mu]m deep cut. Power densities as high as 0.4mW · m⁻², (28mV at open circuit) in the first ever polarization curve for a laminar-flow microbial fuel cell. A model was developed for biofilm behavior incorporating shear and pore pressure as mechanisms for biofilm loss. The model agrees with experimental observations on fluid flow through biofilms, biofilm structure, and other biofilm loss events.
by A-Andrew D. Jones, III.
S.M.

Pharmaceuticals are emerging organic micropollutants that are frequently detected in the aquatic environment. While information on their environmental occurrence is substantial, knowledge gaps exist with respect to their environmental transformation. This licentiate thesis focuses on this research deficit by (1) providing and applying tools for identifying biotransformation products, and (2) investigating the behavior of pharmaceuticals and their transformation products (TPs) in two different experimental systems. Study I established a data-processing method based on peak detection, time-trend filtration and structure assignment, and provides an efficient and reliable way for TP identification. Water/sediment tests were carried out with 9 pharmaceuticals. The method for identifying TPs is based on accurate mass data obtained from high resolution mass spectrometry and a comprehensive data-processing workflow. In total, 16 TPs were identified, 11 of which were confirmed by reference standards. Five of the TPs showed a continuous accumulation over the entire incubation period of 35 days. Study II aimed at studying the influence of water/sediment interactions on the environmental behavior of pharmaceuticals and their TPs. An artificial streaming channel was applied to simultaneously determine the concentrations of parent pharmaceuticals and key TPs in both surface water and sediment pore water under defined hydraulic conditions. All pharmaceuticals dissipated from the test system. The benefit from simultaneously analyzing parent compounds and TPs can be illustrated with carbamazepine, which is generally reported to be persistent. In study II, carbamazepine dissipated continuously from the test system, and the formation of a TP shows that this dissipation can be attributed to microbial biotransformation. The results also indicate that transformation predominantly occurs in the hyporheic zone, but also that TPs can be transported back into the streaming channel. This thesis confirms the crucial role of the hyporheic zone for the elimination of organic micropollutants from rivers and streams. Moreover, the developed workflow for TP identification provides opportunities to efficiently identify TPs for additional micropollutants in laboratory and field studies. In future work, we will study the influence of hydraulic conditions on transformation kinetics and formation of characteristic TPs, and we will extend the work to additional pharmaceuticals. Moreover, we will test the applicability of using TPs as indicators for characterizing ongoing biotransformation in field settings, thereby providing a more efficient way of characterizing the environmental fate of pharmaceuticals.

The unifying concept of endosymbiosis and the ‘holobiont’ is that the interaction of the microbial community and the host’s biology can affect myriad processes from speciation to physiology to behavior. This study explored the role of the microbiome as a potential facilitator of rapid evolution of social behavior in a socially polymorphic species of spider, Anelosimus studiosus. Adult females were collected from solitary and social colonies at two geographically distinct locations, and behaviorally assayed to assign individuals to ‘docile’ or ‘aggressive’ phenotypes. Microbiomes of each individual were analyzed by 16s rRNA sequencing. Correlations were found with external influences on the microbiome (colony type, local environmental microbiota, and among colony), and also between the microbiome and individual’s behavioral phenotype. While causation has not yet been established, these data suggest that demographics and ecology affect the microbiome, and that behavior may be affected by the microbiome.

An investigation into the responses of microbeams to electric actuations is presented. Attention is focused mainly on the use of microbeams in two important MEMS-based devices: capacitive microswitches and resonant microsensors. Nonlinear models are developed to simulate the behavior of the microbeams in each device. The models account for mid-plane stretching, an applied axial load, a DC electrostatic force, and, for the case of resonant sensors, an AC harmonic force. Further, a novel method that uses a reduced-order model is introduced for simulating the behavior of microbeams under a DC electrostatic force. The presented method shows attractive features, like for example, a high stability near the pull-in and a low computational cost. Thus, it can be of significant benefit to the development of MEMS design software. The static behavior of microbeams under electrostatic forces is studied using two methods. One method employs a shooting technique for solving the boundary-value problem that governs the static behavior. The second method is based on solving an algebraic system of equations obtained from the reduced-order model. Further, the eigenvalue problem describing the vibrations of a microbeam around its statically deflected position is solved using a shooting method to obtain the microbeam mode shapes and natural frequencies. The dynamic behavior of resonant microbeams is also investigated. A perturbation method, the method of multiple scales, is used to obtain two first-order nonlinear ordinary-differential equations that describe the amplitude and phase of the response and its stability. The results show that an inaccurate representation of the system nonlinearities may lead to an erroneous prediction of the nonlinear resonance frequency of a microbeam. The case of three-to-one internal resonance between the lowest two modes is treated. Finally, the reduced-order model is used to study the dynamic behavior of the electrostatically actuated microbeams. The proposed models are validated by comparing their results with experimental results available in the literature.
Master of Science

Microbial oxidation of methane has attracted interest as an alternative process for treating landfill gas emissions. Approaches have included enhanced landfill cover layers and biocovers, passive gas drainage and biofiltration, and active gas extraction and biofiltration. Previous research has shown that microbial methane oxidation is affected by a number of factors, many of which are dependent on the environment in which the process is occurring. The aim of this research was to evaluate the behaviour and performance of a passive landfill gas drainage and biofiltration system operating in a temperate climate, and to identify and quantify the factors that determine the behaviour and performance of the system under such conditions. To achieve this a series of field trials were undertaken in Sydney, Australia, over a period of 4 years. The trials were designed to evaluate the effect of a range of factors, including landfill gas loading rate, temperature and moisture content of the biofilter media, biofilter media characteristics, and climatic conditions. The results of the field trials showed that a passive gas drainage and biofiltration system operating in a temperate climate can achieve methane oxidation efficiencies > 90% and that the behaviour and performance of a passive gas drainage and biofiltration system is primarily dependent on 3 factors: the landfill gas loading rate, which varies; the temperature of the biofilter media, which is affected by the temperature of the landfill gas being treated, the level of microbial activity occurring in the biofilter, and local climatic conditions; and the moisture conditions within of the biofilter media, which is affected by local climatic conditions and the characteristics of the biofilter media. Relationships between these factors and the performance of a passive biofilter operating in a temperate climate were developed, where able. A number of design concepts for passive landfill gas drainage and biofiltration were developed. A process for assessing the feasibility of applying the concepts and designing a passive landfill gas drainage and biofiltration system was also developed. In addition, guidelines and recommendations for the design of a passive landfill gas drainage and biofiltration system operating in temperate climate were developed.

The study of Microbial-induced carbonate precipitation (MICP) on organic soil has remained limited. This PhD study intends to fill the knowledge gap by studying MICP of tropical peat. Based on the finding, it was possible (i) to isolate bacteria strains from acidic tropical peat with high urea hydrolysis activities and capable of bio-cementation; (ii) to induce bio-cementation in acidic peat, which leads to strength gain and reduction of permeability; (iii) to improve consolidation behaviour.

Symbiotic microbes are commonly found associated in higher organisms, and often have evolved to play an important role in host biology. Researchers have been studying the effects that microbial symbionts have on host physiology, their effects on host nutrition or protection against pathogenic microbial infections, but have recently begun to examine how they influence the brain and behaviour. Wolbachia pipientis, a gram-negative alpha-proteobacteria, intracellular bacterial symbiont infects approximately 40% of all insect species, including Drosophila. The fruit fly, Drosophila melanogaster, one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, and is naturally infected by several strains of Wolbachia. While Wolbachia are known to infect numerous host tissues, including the brain, little research has focused on how Wolbachia affects the nervous system and behaviour of its host, despite the impact behaviour has on host fitness.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Natural Sciences
Science, Environment, Engineering and Technology
Full Text

Alors que l'épandage de déchets huileux à forte dose sur des sols non cultivés est utilisé pour éliminer les hydrocarbures, les épandages à faible dose sur les terres cultivées ont été peu envisagés. Aussi, des essais ont été conduits au laboratoire et au champ afin de suivre le devenir et les effets, dans le système sol-plante-microorganismes, d'hydrocarbures appliqués à faible dose sous la forme de déblais de forage. Ont été pris en compte la phytotoxicité, le transfert sol-plante, la biodégradation en absence et en présence de plante et l'infiltration des hydrocarbures. Les résultats des études en laboratoire montrent que les hydrocarbures perturbent la germination et la croissance des végétaux. La phytotoxicité dépend de la dose, du type de molécule et varie en fonction des espèces végétales. Aucun transfert d'hydrocarbures dans les parties aériennes du maïs n'est observé. Dans le sol, la majorité des hydrocarbures est dégradée et le résidu final de la biodégradation est constitué de molécules cycliques. En présence de plante, la dégradation est plus rapide et l'exsudation racinaire est un facteur important de stimulation de la dégradation des hydrocarbures dans la rhizosphère. Au champ, l'infiltration sélective des hydrocarbures légers et de certains métabolites dans le profil de sol et dans les eaux de drainage est observée. L’importance du phénomène dépend de la quantité et du type d'hydrocarbures et des conditions pédoclimatiques. L’épandage de déblais de forage en plein champ entraine une légère diminution du rendement des deux premières cultures. Aucun hydrocarbure pétrogénique n'est retrouvé dans les plantes. Dans ces conditions, la vitesse de disparition des hydrocarbures est proportionnelle à la dose d'épandage. Les alcanes linéaires, ramifiés et certains aromatiques sont dégradés alors que les composés cycliques persistent. L’apport de petites quantités de déblais de forage pourrait constituer une alternative au landfarming intensif des déchets huileux. Cependant, la présence de molécules mobiles et toxiques peut avoir des conséquences négatives sur l'agrosystème

The purpose of the research reported in this dissertation is to expand the general knowledge of the chemical properties of monorhamnolipids (produced by P. aeruginosa ATCC 9027) and monorhamnolipid-metal complexes in solution and at interfaces in order to advance the application of these biosurfactants in a variety of applications.The speciation and fragmentation behavior of monorhamnolipids (mRLs) using mass spectrometry at low and high resolution in positive ion mode was investigated as a function of pH, which has yet to be fully discussed to date in the literature. This study laid the groundwork for the speciation and fragmentation behavior of mRLs with two environmentally-relevant heavy metals, Pb²⁺ and UO₂²⁺. It was determined that mRLs form 1:1 and 2:1 mRL-metal complexes with both metal cations across the pH range investigated (pH 4.0, 6.0 and 8.0). mRL-metal complexes were found to fragment differently than free mRLs suggesting coordination of the metal cation in a binding pocket comprised of the mRL carboxylic acid moiety and the rhamnose sugar hydroxyls. This coordination environment was further verified as a function of solution pH using infrared spectroscopy (IR), nuclear magnetic resonance spectrometry (NMR) and hydrogen-deuterium exchange (HDX) mass spectrometry. Adsorption isotherms for mRLs and mRL-metal complexes on two soil components, silica and goethite, were characterized as a function of solution pH using ATR-FTIR and successfully fit to the Frumkin-Fowler-Guggenhiem isotherm to extract relevant thermodynamic adsorption parameters. These studies showed that at low pH, mRLs form bilayers on these surfaces, but the adsorption affinity of the mRLs is dictated by the molecular interactions these species have with the specific oxide surface. At neutral and basic pH values, mRLs were found to adsorb to silica despite the fact that both the surface and the mRLs are negatively charged. The Lewis acid/base interactions of mRLs with goethite at neutral and basic pH values results in multilayer adsorption. Adsorption of mRL-metal complexes on silica and goethite suggests that the interactions between these complexes and soil surfaces may have a direct impact on metal ion remediation efficiencies using mRLs. The adsorption affinity of mRL-Pb²⁺ complexes to silica is greater than that of mRL-UO₂²⁺ complexes; however, mRL-UO₂²⁺ complexes precipitate at the interface. The adsorption affinity of mRL-metal complexes on goethite is not significantly lower than for free mRLs; however, the surface coverage decreases.

Pre-vegetation alluvium is unique; at the present day, plants affect multiple aspects of river functioning and deposition and so those rivers that operated before the evolution of land plants largely lack modern sedimentological analogue. However, such rivers were the norm for the first 90% of Earth history and so a better understanding of their sedimentary product enables insight into both the fundamental underlying mechanisms of river behaviour and the ways in which fluvial processes operated on ancient Earth and other rocky planets. This study presents five original fieldwork based case studies and an analysis of a holistic database of all of Earth’s pre-vegetation alluvium. Together these research strands offer perspectives on the sedimentological characteristics and stratigraphic trends of pre-vegetation alluvium and the behaviour and functioning of pre-vegetation rivers. Results show that, in pre-vegetation alluvial settings: 1) a variety of fluvial styles are represented, but diminished in comparison with syn-vegetation alluvium; 2) ‘sheet-braided’ architectures are common but may record a variety of fluvial planforms; 3) meandering planforms were less frequent, particularly in small- to moderate-sized river systems; 4) mudrock is on average 1.4 orders of magnitude less common than it is in syn-vegetation alluvium; and 5) microbial matgrounds were present, but had negligible effect on preserved architecture and facies. This thesis demonstrates that whilst the physical laws governing fluvial fluid-sediment interaction have not changed, the theatre in which they operated irrevocably evolved with the greening of the continents.

Le fipronil, molecule insecticide appliquee en traitement de pelliculage sur les semences de mais, penetre directement, via un flux transtegumentaire ou transpedicellaire, dans l'albumen et le scutellum. Un taux maximal de 7% de la quantite appliquee penetre dans la graine des la phase de pelliculage. La penetration transtegumentaire dans la graine redemarre ensuite lors de la phase de gonflement et de germination de la semence, pour atteindre 20% de la quantite initiale apportee par le pelliculage, dans la plantule, apres 6 jours de croissance. Pour les parties neoformees du mais, c'est l'eau du sol qui devient le vecteur du fipronil (apres solubilisation a partir du pelliculage). Cette eau, chargee en fipronil, conduit aussi a un chargement du complexe argilo-humique dans la zone de la graine. Dans la plante de mais, comme l'ont montre les etudes realisees par rhone-poulenc ainsi que nos observations au sein d'un agrosysteme simplifie, le fipronil est partiellement metabolise. Compte tenu de la longue periode (5 mois) entre le traitement et la fructification du mais et des aptitudes a la degradation que presentent cette espece, pour donner des metabolites relativement lipophiles, il est peu probable de trouver des residus importants dans les grains. La larve de taupin, ravageur souterrain des cultures de mais et organisme cible du fipronil, acquiert cette matiere active principalement par ingestion. Nous avons demontre que, pour un plant de mais, ce sont les reserves de la graine qui sont les plus appetentes pour le taupin et qu'elles sont consommees. Pour les atteindre, les larves consomment pelliculage et teguments et acquierent ainsi la quantite de fipronil declenchant la letalite. La quantite de fipronil absorbe par le taupin, par voie transtegumentaire, a ete determinee comme etant faible, et nous n'avons pas mis en evidence de metabolisation du produit au sein de la larve. Au niveau du sol, le fipronil est en equilibre de partition entre l'eau du sol et le c omplexe argilo-humique, sur lequel il va s'adsorber. Deux elements importants dans la dissipation du fipronil sont les vers de terre et la biomasse microbienne. La biomasse microbienne acquiert le fipronil a la mesure de sa partition avec l'eau du sol. Cette flore bacterienne est susceptible de metaboliser cette matiere active (resultats presentes dans le dossier d'homologation). Parallelement, les vers de terre sont potentiellement susceptibles de concentrer des quantites importantes de fipronil, et de le metaboliser en un produit majoritaire gardant des proprietes insecticides. Cependant, le ver ne manifeste aucune atteinte toxique, ce qui tend fortement a demontrer que ses canaux chlore ne sont pas atteints par le fipronil, contrairement a ceux des insectes. Le ver apparait aussi comme un acteur potentiellement important de la chaine de dissipation du fipronil. Nous avons etudie en detail le processus de partition du fipronil entre l'eau et divers etres vivants (differents organes du mais, taupins, micro-organismes, vers de terre). L'equilibre de partition analyse et mesure est une fonction non seulement de la teneur en lipides libres des etres ou organes etudies, mais aussi de la presence de proteines a sites lipophiles comme la zeine ou de polymeres lipophiles comme la lignine. Dans l'agrosysteme simplifie etudie au laboratoire en conditions controlees, il semble que l'efficacite anti-taupin depende directement de la presence du fipronil dans les reserves des graines ou associe aux reserves lors de l'ingestion. Le fipronil contenu dans les organes neoformes ou adsorbe sur le complexe argilo-humique ne semblent jouer qu'un role mineur du point de vue de l'efficacite insecticide. L'apport du fipronil par le biais du traitement de semences sur mais, dans la lutte anti-taupin, presente des caracteristiques tres interessantes d'un point de vue environnemental. La surface de sol traitee est faible, la matiere active presente une faible mobilite dans le sol et la position enfouie du fipronil autour des semences ecarte la possibilite d'une contamination atmospherique. Des modifications de la formulation utilisee en traitement de semences sont suggerees pour augmenter la penetration de la matiere active dans l'albumen lors de la phase de pelliculage des semences. Ceci pourrait conduire a une diminution de la quantite necessaire de fipronil a l'hectare, tout en gardant l'efficacite agronomique, en raison d'une diminution de la quantite liee au sol. Evidemment, le succes d'une telle modification necessiterait l'absence de phytotoxicite au niveau de la semence et de la plantule.

Silver nanoparticles (AgNPs) are tiny particles of silver with nanoscale dimensions (between 1 and 100 nm) and unique antimicrobial properties. AgNPs are potential environmental contaminants increasingly applied in consumer products. The effects on nontarget biological systems are not clearly defined. Research has shown that AgNPs may inhibit the function of bacteria responsible for organic matter decomposition, nutrient cycling, and control of pathogens population in the aquatic system. AgNPs have recently been detected in a treated municipal wastewater raising concerns about their potential risk to aquatic organisms. The microbial community in the sediment has a greater risk of AgNPs exposure, as metals in aquatic systems settle in the sediment. Studies have shown that microbial community growth and carbon sources utilization patterns were altered in response to AgNPs exposure in marine estuarine sediments. The antimicrobial activity of AgNPs in freshwater sediments may be different due to the water chemistry. Few studies have evaluated the toxicity of AgNPs in freshwater sediments due to the complex nature of their water chemistry. The current study investigated microbial community’s responses to AgNPs in sediments collected from a local stream. Microbial growth and activity assays were performed to determine whether AgNPs pose a risk to the microbial community in freshwater sediments. We found that AgNPs inhibited microbial growth, enzyme activity, and catabolic capabilities (P < 0.05). The number of viable bacterial cells and the ability of the microbial community to utilize different carbon sources decreased at 0.431 and 0.538 mg AgNPs kg-1 sediment, which are found within the estimated AgNPs concentration range in sediments. AgNPs inhibited the activity of glucosidase, an enzyme responsible for carbohydrate metabolism, but the activity of alkaline phosphatase was not affected. The current study demonstrates that AgNPs can inhibit the growth and functional diversity of beneficial microorganisms, which may affect the quality of surface waters and their designated uses. These adverse effects are expected due to the demonstrated antimicrobial properties of AgNPs incorporated in several commercial products. Toxicological data generated from this study could be incorporated in ecological risk assessment by regulatory agencies to assess the impacts of AgNPs on ecosystem systems.

Il moscerino asiatico (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), originario dell'Asia orientale, è uno dei principali parassiti emergenti di colture pregiate in Europa e nelle Americhe. Nel 2008, si è verificata una rapida invasione di questa specie di fitofago dei frutti rossi in Europa e nelle Americhe. Di conseguenza, D. suzukii è attualmente uno degli insetti dannosi più rilevanti di colture pregiate, attaccando i frutti rossi e l'uva da vino, e causando milioni di dollari di danni ogni anno. A differenza delle altre Drosophilae, la SWD è in grado di penetrare la buccia della frutta in maturazione e di deporre le uova al suo interno, dove gli stadi larvali si nutrono, si sviluppano e causano danni. Questo piccolo moscerino della frutta, una specie strettamente imparentata con D. melanogaster e D. simulans, ha sviluppato un sofisticato sistema olfattivo che può rilevare l'odore della frutta e altri odori provenienti da un habitat potenzialmente adatto. Tali composti volatili sono mediatori a lungo raggio del suo comportamento. Lo studio del sistema olfattivo di D. suzukii può contribuire quindi a migliorare la nostra comprensione del comportamento e della fisiologia di questi insetti e ci permetterà di sviluppare soluzioni efficaci di controllo dei parassiti. Le attuali strategie di controllo si basano sull'uso massiccio di insetticidi, che hanno un impatto ecologico negativo e a lungo termine non sono né efficaci né sostenibili. Gli strumenti che l'ecologia chimica fornisce si adattano perfettamente ai programmi di gestione integrata (IPM) di D. suzukii e potrebbero offrire un approccio alternativo e più sostenibile per limitarne la diffusione e i danni. In questa tesi di dottorato, abbiamo usato nelle nostre indagini saggi microbiologici, chimici, elettrofisiologici di laboratorio e studi in campo aperto. L'obiettivo generale di questo studio di dottorato è stato quello di indagare il potenziale di una miscela di vino, aceto di mele e canna da zucchero insieme a composti volatili microbici in una trappola innovativa progettata per migliorare l'azione attrattiva nei confronti di D. suzukii in campo aperto. Approcci sostenibili per limitare la diffusione di D. suzukii e i danni che causa richiedono sempre esche e trappole efficaci. Il nostro obiettivo quindi è stato quello di sviluppare un'esca innovativa ed efficace da implementare in un sistema di trappole selettive per il controllo di SWD come parte dei programmi di gestione integrata dei parassiti (IPM) in campo aperto. Mentre la maggior parte delle ricerche su D. suzukii si è concentrata su composti volatili emessi dalla frutta o dai lieviti presenti sui frutti ospiti, c’erano poche prove su come altri microrganismi che emettono composti volatili possano giocare un ruolo nel comportamento di questo insetto. Inoltre, era poco conosciuto il ruolo di composti volatili batterici nel comportamento di ricerca degli ospiti di SWD e se quindi essi possano essere applicati per migliorare il controllo ed il monitoraggio di questa specie invasiva. Abbiamo usato Droskidrink®, un prodotto commerciale per la cattura di D. suzukii, come esca di base per il miglioramento e ulteriori indagini. Abbiamo dimostrato che l'aggiunta di batteri lattici al Droskidrink® nella prima settimana dopo la fermentazione migliora l'attrattività verso SWD. Abbiamo studiato i composti volatili chiave per SWD, emessi durante il processo di fermentazione del vino e dell'aceto mediato da batteri lattici. Inoltre, abbiamo trovato una relazione tra gli stessi composti volatili in grado di mediare il comportamento di D. suzukii e altri organismi a diversi livelli trofici. In particolare, abbiamo usato prodotti metabolici di Saccharomyces cerevisiae e il noto endoparassitoide di SWD Trichopria drosophilae Perkins (Hymenoptera; Diapriidae). Nella prima parte di questa tesi di dottorato (Capitolo 2), abbiamo studiato l'uso dei batteri come bio-catalizzatori dei processi metabolici che avvengono durante la fermentazione malolattica di una miscela di vino-aceto di mele-canna da zucchero attrattivo per D. suzukii. Abbiamo prima valutato l'attrattività dell'esca alimentare Droskidrink® integrata con diversi ceppi di batteri lattici. Questo esperimento è stato condotto in pieno campo in un vigneto commerciale. Abbiamo usato Droso-Trap® Biobest, e Droskidrink® integrato con Oenococcus oeni, Pediococcus spp e Lactobacillus spp. Inoltre, il rendimento dei ceppi batterici attrattivi è stato studiato in condizioni di laboratorio. Successivamente, abbiamo studiato la risposta elettroantennografica di SWD ai ceppi di O. oeni più attrattivi, integrati con Droskidrink®. I risultati hanno mostrato che dei diversi batteri lattici studiati negli esperimenti di laboratorio e sul campo, tre ceppi di O. oeni erano i più attivi nei confronti di D. suzukii. Nel capitolo 3, abbiamo eseguito l'estrazione dei composti volatili da miscele ottenute con diversi ceppi di O. oeni aggiunti al Droskidrink® per valutare come i composti volatili emessi dai batteri influenzassero la composizione chimica del Droskidrink®. Abbiamo testato l'influenza dei batteri lattici (due ceppi di O. oeni precedentemente selezionati come i ceppi più interessanti nelle prove sul campo) e la successiva fermentazione malolattica nelle miscele vino-aceto-zucchero, per un periodo di tre settimane. Per l'estrazione dei composti volatili, sono state utilizzate due diverse metodologie di estrazione, ovvero la raccolta diretta dallo spazio di testa e il metodo Closed-Loop-Stripping-Analysis (CLSA). La fermentazione è stata impostata in modo tale che i metaboliti sono stati estratti una, due e tre settimane dopo l'inizio della fermentazione malolattica. Per l'identificazione chimica dei composti altamente volatili, abbiamo utilizzato l'analisi diretta dello spazio di testa collegata a un gascromatografo con un rivelatore selettivo di massa. I composti volatili estratti nel solvente sono stati analizzati in un sistema GC-MS standard su due diversi tipi di colonna per aumentare il numero di composti identificati. Inoltre, sono stati utilizzati standard chimici sintetici per la co-iniezione e la conferma chimica. Successivamente, abbiamo studiato la risposta elettroantennografica dei composti volatili raccolti, dissolti in un solvente, su SWD femmina. L'esperimento comportamentale a scelta multipla è stato eseguito in condizioni di laboratorio e per sostenere la nostra ipotesi è stato anche testato in studi in campo aperto con l'uso di un innovativo sistema di cattura. I risultati hanno rivelato che la fermentazione malolattica con specifici ceppi di batteri lattici ha prodotto con una composizione specifica di composti volatili che ha reso la nostra miscela più attrattiva per D. suzukii. I nostri risultati hanno descritto la composizione chimica delle miscele emesse dal Droskidrink® dopo la fermentazione batterica. Questi composti volatili includevano alcuni nuovi composti elettrofisiologicamente attivi per SWD, come l'eugenolo e la triacetina. Inoltre, i risultati hanno mostrato una vasta gamma di composti organici volatili diversi che hanno fortemente influenzato il comportamento di SWD. È interessante notare che l'innovativa trappola progettata utilizzando solo 15 mL di una miscela di vino-aceto-zucchero di canna con un ceppo beta attrattivo di O. oeni ha aumentato di due volte la cattura della trappola quando è stata saggiata rispetto a un diverso attrattivo disponibile in commercio, ovvero Scentry® (miscela brevettata, Scentry Biologicals Inc., Billings, MT, USA). I risultati hanno ulteriormente confermato la teoria comunemente accettata dell'importanza dei composti volatili ubiquitari delle piante nell'attrarre gli insetti fitofagi. Nonostante una maggiore comprensione del ruolo dei semiochimici per manipolare il comportamento di SWD, attualmente le tecniche basate su tali composti in campo aperto non sono ben consolidate per questa specie invasiva. Inoltre, la non selettività nella cattura di D. suzukii diminuisce l'efficacia dei sistemi di trappole sviluppati. Pertanto, nel Capitolo 4, abbiamo mirato a identificare composti specifici che possono repellere altre specie di drosofila e aiutare nella costruzione di sistemi di cattura più selettivi. Usando la gascromatografia-spettrometria di massa GC-MS, una combinazione di gascromatografia-elettroantennografia GC-EAD, e biosaggi in gabbia a scelta multipla con composti volatili sintetici, abbiamo cercato di trovare un composto repellente per le Drosophile catturate usando l'attuale sistema di trappola. Abbiamo condotto la nostra ricerca sulla specie sorella di D. suzukii, Drosophila simulans Sturtevant. Successivamente, è stata selezionata la miscela più promettente di composti putativamente repellenti. I risultati hanno rivelato che diverse miscele di composti erano significativamente non attrattivi a causa della presenza di composti repellenti. Nel complesso, questi risultati indicano che i composti: benzaldeide, eugenolo, etanolo, etile isovalerato, feniletil acetato, isoamil lattato, 1-octen-3-olo, etile caprolete, limonene, p-cimene, acido valerico erano significativamente repellenti. Infine, l'uso di pesticidi tossici per combattere le specie invasive deve essere ridotto. I prodotti chimici non ecologici danneggiano gravemente l'ambiente. Sono stati usati insetticidi dannosi e tossici, non solo per la salute umana ma anche per tutti gli organismi nell'habitat. Le sostanze chimiche tossiche ostacolano i nemici naturali degli insetti nocivi, i parassitoidi e i predatori. La gestione integrata dei parassiti (IPM) mira a bilanciare l'uso di buone pratiche agricole con la pianificazione strategica, il monitoraggio precoce, il controllo biologico e molte pratiche agricole diverse con un uso minimo di composti chimici che hanno dimostrato di essere pericolosi per la biodiversità. Il controllo biologico con l'uso di nemici naturali è una parte importante dell'IPM, in combinazione con altri metodi, ad esempio un buon monitoraggio e la cattura massale. Applicare molte strategie diverse allo stesso tempo potrebbe portare al raggiungimento dell'obiettivo. Proteggere e promuovere gli agenti di biocontrollo naturalmente presenti in un agroecosistema è quindi fondamentale (lotta biologica conservativa) insieme al rilascio razionale di quelli commercialmente disponibili (lotta biologica aumentativa). Comprendere il comportamento dei nemici naturali e la loro scelta degli insetti ospiti è quindi uno dei passi chiave nel miglioramento della lotta biologica. Il comportamento dei nemici naturali è determinato da composti chimici rilasciati nell'ambiente dagli insetti ospiti, dalle piante e dalla fonte di cibo dell'insetto ospite. I composti chimici provenienti dalla pianta ospite sono stati ampiamente studiati. Recentemente, l'attenzione si è concentrata sulle sostanze che sono prodotte durante il metabolismo dei microrganismi. Questi composti chimici sono chiamati composti organici volatili microbici, mVOCs. I mVOCs possono mediare il comportamento degli insetti e portare alla scelta dei siti di accoppiamento, ovodeposizione e alimentazione. Si sa poco su come i mVOCs influenzino il comportamento dei nemici naturali e sulla loro applicazione nell'IPM. Pertanto, nel capitolo 5 abbiamo cercato di studiare uno dei principali endoparassitoidi di D. suzukii, Trichopria drosophilae e il suo comportamento nel contesto della ricerca dell'ospite. In questo capitolo, abbiamo studiato il comportamento di ricerca dell'ospite e l'utilizzo degli stessi segnali volatili nella Drosophila e nel suo parassitoide, emessi da una delle fonti primarie della dieta ricca di proteine della drosofila, Saccharomyces cerevisiae. Nel complesso, questa tesi di dottorato ha fornito una migliore comprensione dell'interazione mediata da composti volatili tra i microrganismi responsabili della fermentazione di un attrattivo comune per D. suzukii, una miscela di vino-aceto-zucchero di canna. Dagli studi neurofisiologici, microbiologici e chimici, e attraverso studi comportamentali di laboratorio e di campo, abbiamo ottenuto importanti risultati innovativi. Queste conoscenze possono essere sfruttate per sviluppare un nuovo strumento per il monitoraggio di D. suzukii all'inizio del suo movimento nei campi agricoli dalle aree di rifugio invernali, e anche per la cattura massale durante il periodo di massima infestazione in estate. Inoltre, i risultati del nostro studio possono portare all’ottimizzazione dell’attuale gestione integrata di SWD e di conseguenza a pratiche più sostenibili per la gestione di le specie di insetti invasive. Abbiamo utilizzato i semiochimici specifici emessi da microrganismi legati alle piante ospiti dell’insetto per aumentare l'attrazione e come base per la progettazione di trappole innovative.
The spotted-wing Drosophila (SWD), Drosophila suzukii Matsumura (Diptera: Drosophilidae), native to Eastern Asia, is one of the main emerging pests of valuable crops in Europe and the Americas. In 2008, rapid invasion of this soft fruit pest species occurred across Europe and the Americas. Consequentially, D. suzukii is currently one of the most relevant pest of valued horticultural crops, attacking soft fruit and wine grapes, and causing millions of dollars of damage annually. In contrast with other Drosophilae, SWD is capable of penetrating the skin of ripening fruit and laying eggs inside, where larval instars feed, develop and cause damage. This small fruit fly, a closely-related species of D. melanogaster and D. simulans, has developed a sophisticated olfactory system that can detect fruit odour and other odours coming from a potentially suitable habitat. Volatile cues are long-range mediators of its behaviour. Investigation of the olfactory system of D. suzukii will thus develops our understanding of the behaviour and physiology of these insects and allows us to develop effective pest control solutions. Current control strategies rely on the heavy use of insecticides, which have a negative ecological impact, and in the long run are neither effective nor sustainable. The tools that chemical ecology provides fit perfectly into D. suzukii integrated pest management (IPM) programmes, and could offer an alternative, more sustainable approach to limit its spread and damage. In this PhD thesis, we used microbiological, chemical, electrophysiological and laboratory bioassays and open field studies in our investigations. The overall aim of this PhD study was to investigate the potential of a tailored wine-apple cider-sugar cane mixture together with microbial volatiles in an innovative trap designed to improve the attractiveness of D. suzukii in the open field. Sustainable approaches to limit the spread of D. suzukii and the damage it causes always require effective lures and traps. We, therefore, intended to develop an innovative and effective lure to be implemented in a selective trapping system for controlling SWD as part of integrated pest management (IPM) programmes in open fields. While most research investigating D. suzukii has focused on volatile cues derived from host fruit or yeast, there is little evidence about how other microorganisms emitting volatile compounds could play a role in this fly’s behaviour. Moreover, little is known about how bacterial microbial volatiles affects the behaviour of SWD and whether they can be applied to improve integrated pest management control of this invasive species. We used Droskidrink®, a commercial product for catching D. suzukii, as a basic lure for improvement and further investigations. We demonstrated that adding lactic acid bacteria to Droskidrink® in the first week after fermentation improves attractiveness for SWD. We investigated key odourant cues for SWD, emitted by the wine-vinegar-lactic acid bacteria fermentation process. Furthermore, we found a connection between the same volatile cues capable of mediating the behaviour of Drosophila suzukii and other organisms on different trophic levels. For investigation across different trophic levels, we used Saccharomyces cerevisiae metabolic products and the well-known D. suzukii endoparasitoid Trichopria drosophilae Perkins (Hymenoptera; Diapriidae). Volatiles extracted from different sources are used to develop simple attractants with the use of a small number of compounds in a special ratio and concentration. Currently, the most prominent lures contain volatiles isolated from Merlot wine, rice vinegar, wine vinegar, apple cider vinegar, apple juice, fermented apple juice, the surface of raspberries or crushed berry fruits, including blueberries, cherries and strawberries. Some volatile compounds are isolated from acetic acid bacteria grown in different liquid media, and volatiles from different yeast fermentations. In the first part of the PhD study (Chapter 2), we investigated the use of bacteria as a bio-catalyser of metabolic processes occurring during malolactic fermentation of a wine-apple cider-sugar cane mixture attractive to Drosophila suzukii. We first evaluated the attractiveness of Droskidrink® food bait supplemented with different lactic acid bacteria strains. This experiment was conducted in open field studies in a commercial vineyard. We used Droso-Trap® Biobest, and Droskidrink® supplemented with Oenococcus oeni, Pediococcus spp and Lactobacillus spp. Moreover, the performance of attractive bacterial strains was investigated under laboratory conditions. Next, we studied the electroantennography response of SWD flies to the most attractive O. oeni strains, supplemented with Droskidrink®. The results showed that of the different lactic acid bacteria studied in laboratory and field experiments, three strains of O. oeni were most active to Drosophila suzukii. In Chapter 3, we performed volatile extraction of the mixtures, with different O. oeni strains added to Droskidrink® to assess how the volatile compounds emitted by bacteria affected the chemical composition of Droskidrink®. We tested the influence of lactic acid bacteria (two strains of O oeni previously selected as the most attractive strains in the field trials) and subsequent malolactic fermentation in wine-vinegar-sugarcane mixtures, over a period of three weeks. For volatile extraction, two different extraction methodologies were used, namely Direct Headspace Collection and the Closed-Loop-Stripping-Analysis (CLSA) method. Fermentation was set up in such a way that volatiles were extracted one, two and three weeks after the beginning of malolactic fermentation. For chemical identification of highly volatile compounds, we used direct head-space analysis connected to a Gas-Chromatograph with a Mass Selective Detector. Volatile extracts in the solvent were analysed in a standard GC-MS system on two different types of column to increase the number of identified compounds. Moreover, synthetic chemical standards were used for co-injection and chemical confirmation. Next, we studied the electroantennographical response of the collected volatiles, dissolved in a solvent, on female SWD flies. The behavioural multi-choice experiment was performed under laboratory conditions and to support our hypothesis was also tested in open field studies with the use of an innovative trapping system. The results revealed that malolactic fermentation with specific LAB strains tuned VOC composition in a way that made our tested mixture more attractive to D. suzukii. Our results revealed the chemical composition of various volatiles emitted by Droskidrink® after bacterial fermentation. These volatiles included some newly electrophysiologically-active compounds for SWD, such as eugenol and triacetin. Additionally, the results showed a wide range of diverse volatile organic compounds that strongly mediated the behaviour of SWD. Interestingly, the field innovative trap designed using just 15 mL of a mixture of wine–vinegar-sugar cane with an attractive beta strain of O. oeni increased trap catch two-fold when tested compared to a different commercially available attractant, namely Scentry® (proprietary blend, Scentry Biologicals Inc., Billings, MT, USA). The results further confirmed the commonly accepted theory of the importance of ubiquitous plant volatiles in attracting insects. Despite an increased understanding of the role of volatile emission as insect semiochemicals, and their use to manipulate SWD behaviour, at present semiochemically-based techniques in the open field are not well-established for this invasive species. Furthermore, non-selectivity and spillover in the catching of D. suzukii decreases the effectiveness of the trap systems developed. Therefore, in Chapter 4, we aimed to identify specific compounds that may repel other drosophila species and help in building more selective trapping systems. Using gas chromatography-mass spectrometry GC-MS, a combination of gas chromatography-electroantennography GC-EAD, and multi-choice cage bioassays with synthetic volatile compounds, we tried to find a repellent compound for untargeted Drosophilae caught using the current trap system. We conducted our research on the Drosophila suzukii sister species Drosophila simulans Sturtevant. Next, the most promising mixture of putatively repellent compounds was selected. The results revealed several compound mixtures were significantly not attractive because of repellent compound presence. Overall, these results indicate that compounds: benzaldehyde, eugenol, ethanol, ethyl isovalerate, phenylethyl acetate, isoamyl lactate, 1-octen-3-ol, ethyl caprolete, limonene, p-cymene, valeric acid were significantly repellent. Finally, the use of toxic pesticides to fight invasive species must be reduced. Environmentally unfriendly chemicals severely damage the environment. Insecticides that are harmful and toxic, not just for human health but also for all organisms in the habitat, have been used. Toxic chemicals impede naturally occurring enemies of pest insects, parasitoids and parasites. Integrated pest management (IPM) aims to balance the use of good agricultural practices with strategic planning, early monitoring, biological control and many different agricultural practices with minimal use of chemical compounds that have been proven to be dangerous for bio-diversity. Biological control using natural enemies is an important part of IPM, not just one aspect and strategy, as good monitoring and mass trapping could lead to a decline in the numbers of SWD in agricultural fields. Applying many different strategies at the same time could lead to achievement of the goal. Supplying the agro-environment with biological pest control (BPC) by boosting the natural population of parasitoids and predators is significant. In biological control (BC), naturally present beneficial organisms are supported with commercially reared natural enemies. One of the challenges in biological control is to maintain and attract beneficial insects to orchards (agricultural fields). Understanding the behaviour of natural enemies and their choice of host insects is one of the key steps in improvement of BC. The behaviour of natural enemies is determined by chemical cues released in the environment by host insects, plants, and the food source of the host insect. Chemical cues originating from the host plant have been widely studied. Recently, attention has been focused on chemical cues that are produced as products of the microorganism’s metabolism. These chemical cues are called microbial volatile organic compounds, mVOCs. mVOCs can mediate insect behaviour and lead to the choice of the mating, oviposition and feeding sites. Little is known about how mVOCs influence the behaviour of natural enemies, and their application in IPM. Therefore, in Chapter 5 we aimed to investigate one of main Drosophila suzukii endoparasitoids, Trichopria drosophilae and its behaviour in the context of host searching. In this chapter, we investigated host searching behaviour and utilisation of the same volatile cues in Drosophila and its parasitoid, emitted by one of the primary drosophila protein-rich diet sources, Saccharomyces cerevisiae. Overall, this PhD study has provided a better understanding of volatile mediated interaction between microorganisms and fermentative effects on a worldwide homemade attractant for Drosophila suzukii, a mixture of wine-vinegar-sugar cane. From fundamental neurophysiological, microbiological and chemical studies, through laboratory insect behavioural studies applied to open field studies, we have obtained important findings. This knowledge, combined with applied studies in the open field, may be exploited to develop a novel tool that detects D. suzukii at the beginning of its movement to agricultural fields from winter shelter areas, and also for mass trapping during the peak infestation period, when farmers have not intervened during the bottleneck period of arrival of SWD in the field. Moreover, the results of our study potentially lead to improved integrated management control of SWD and consequently to more sustainable practices in dealing with invasive insect species. We utilised microbe host-specific semiochemicals for attraction and as a basis for innovative trap design.

El biochar, el carbón especial obtenido a través del proceso de pirólisis de la biomasa, se ha propuesto como un medio de captura de carbono a través del suelo. También se ha considerado útil para mejorar algunas de las propiedades pertinentes del suelo y para reducir los efectos adversos de los contaminantes. Pero el uso de biochar debe garantizar, además de su conservación, que no genere efectos adversos en los organismos vivos, en las funciones ecosistémicas del suelo, o sobre los sistemas naturales adyacentes. Esta tesis pretende contribuir al conocimiento en este sentido, mediante el estudio de: -La capacidad del biochar para proteger la materia orgánica más lábil del suelo; -Los efectos adversos causados por el biochar una vez incorporado en el suelo, en función del tipo de proceso de pirólisis y las dosis aplicadas; y -La modulación de los efectos secundarios tóxicos de algunos pesticidas cuando se incorporan a los suelos que han recibido un aporte de biochar Para alcanzar estos objetivos, se utilizaron tres tipos de biochar producidos a partir de la misma biomasa (astillas de madera de pino) como enmienda del suelo: biochar producido por pirólisis lenta (PL), el obtenido por pirólisis rápida (PR) y el obtenido a través de la gasificación de la biomasa (PG). La adición de biochar genera cambios en la estructura y la biología del suelo, sea por la modificación positiva o negativa de su equilibrio físico y biológico. Considerando que la comunidad microbiana juega un importante papel regulador en el suelo, condicionando las reacciones bióticas más importantes, la biomasa microbiana y su actividad respiratoria se utilizaron como indicadores de los efectos producidos por la adición de biochar. Se han usado modelos de adsorción que se han utilizado para evaluar la capacidad del biochar para adsorber y proteger la materia orgánica más lábil del suelo. Se disenó un experimento factorial completo para comprobar los efectos de tres factores individuales (biochar, nutrientes y adición de glucosa) y su relación con la mineralización de la materia orgánica del suelo. Los resultados de este trabajo indicaron que cada tipo de biocarbón estableció diferentes interacciones con el suelo. El ensayo ecotoxicológico sugirió que el biochar de pirólisis rápida (PR) es el menos recomendado si se utiliza en dosis superiores a la ED50 calculada. No se obtuvo evidencia de interacciones de protección de materia orgánica lábil, como la glucosa, por nuestros resultados. Aunque la glucosa fue absorbida con eficacia en el suelo tratado con biochar, la absorción no actuó como un mecanismo de protección a largo plazo contra la mineralización. El biochar producido por la pirólisis lenta o por gasificación, PL y PG, resultaron los más recomendables desde el punto de vista de la respuesta de la biomasa microbiana. Sobre la base de estos resultados, PL fue el biochar elegido para evaluar si la aplicación de tres plaguicidas podía tener efectos adversos detectables sobre la actividad microbiana del suelo, y para evaluar si la adición de biochar modifica la toxicidad o los efectos adversos de estas sustancias químicas en el suelo. La modulación de los efectos producidos por estas sustancias químicas ha quedado demostrada. Estos resultados remarcan cómo diferentes biochars ejercen diferentes interacciones con el suelo, en función de sus características físico-químicas intrínsecas. En esta tesis se hace hincapié en la importancia de futuros trabajos centrados en la formulación de directrices para la aplicación de biochar en el suelo.
Biochar, the particular charcoal obtained through the process of pyrolysis of biomass, has been proposed as a mean of carbon sequestration through soil. It has been also considered useful to improve some relevant properties of the soil fertility and to reduce adverse effects of pollutants. But the use of biochar must ensure, in addition to its conservation, that it does not generate adverse effects on living organisms, on the soil ecosistemic functions, or on the adjacent natural systems. This thesis attempts to contribute to knowledge in this regard, by studying: - The biochar's ability to protect the most labile soil organic matter; - The hypothetic adverse effects caused by biochar once incorporated into the soil, depending on the type of pyrolysis process and doses applied ; and - The modulation of the toxic side effects of some pesticides when incorporated into soils that have received a provision of biochar. To achieve these goals three types of biochars produced from the same starting biomass (pine wood chips) were used to amend soil: biochar produced by slow pyrolysis (PL), biochar obtained from fast pyrolysis (PR) and biochar obtained through the gasification of biomass (PG). The addition of biochar results in changes of the structure and biology of the soil, then in positive or negative modification of its physical and biological balance. Considering that microbial community plays a major regulatory role in the soil, conditioning the most important biotic reactions, microbial biomass and soil respiration were used as indicators of the effects produced by the addition of biochar into the soil. Sorption models were used to assess the ability of biochar to adsorb and protect the most labile soil organic matter. A fully factorial experiment was designed to check the effects of three single factors (biochar, nutrients and glucose addition) and their relationship with the whole SOM mineralization. The results of this work demonstrated that each kind of biochar established different interactions with the soil.

A leishmaniose visceral, na sua forma clinica ativa, caracteriza-se por febre de longa duração, hepatoesplenomegalia e caquexia. No Brasil, a letalidade é, em média, de 7% e as principais causas de morte são: hemorragia, comorbidade com doenças imunossupressoras e infecção bacteriana. O mecanismo de aumento de infecção bacteriana na LV não está claro e uma das hipóteses, é que pode haver translocação bacteriana da mucosa intestinal para o lúmen dos vasos sanguíneos e ocasionar uma maior severidade da resposta imuno-inflamatória e com consequente piora clínica. O objetivo deste trabalho foi avaliar a ocorrência de translocação microbiana em hamsteres infectados experimentalmente com Leishmania (L.) infantum e correlacionar com as alterações histopatológicas encontradas no intestino dos animais infectados. Hamsteres (Mesocricetus auratus) foram infectados intraperitonealmente com 2x107 amastigotas de L. (L.) infantum e eutanasiados após 48, 72 horas e 15, 45 e 90 dias de infecção. Como grupo controle foram utilizados hamsteres inoculados intraperitonealmente com meio de cultura RPMI. Foram coletados: sangue, fezes, baço, intestinos grosso e delgado. Para detecção de amastigotas na mucosa intestinal, foi utilizada a técnica de PCR em tempo real (qPCR), imunohistoquímica e análise histopatológica, sendo que nesta técnica também foram avaliadas alterações histológicas no tecido intestinal. O baço foi utilizado para determinar a carga parasitária através da técnica de Stauber. Para detecção da translocação microbiana ou produtos desde, foi realizada a quantificação de lipopolissacarídeo (LPS) em plasma. Para avaliar a possível mudança da flora bacteriana intestinal, foi realizado sequenciamento bacteriano de amostra de fezes de hamsteres controles e infectados nos vários tempos de infecção. Observamos aumento da carga parasitária em baço e em intestino com o decorrer da infecção, sendo a diferença significativa aos 90 dias de infecção. Paralelamente, observamos aumento de LPS circulante nos animais infectados em diferentes tempos, 48 horas, 72 horas, 45 dias e 90 dias, com diminuição no período intermediário de 15 dias, porém com diferença significante somente aos 90 dias após a infecção em relação ao grupo controle. Alterações histopatológicas foram observadas no intestino grosso e delgado, variando de infiltrado inflamatório leve a grave, enterite, histiocitose e ainda presença de amastigotas. As alterações observadas ocorreram a partir de 48 horas de infecção, diferenciando a população do infiltrado inflamatório entre neutrófilos, linfócitos, e ainda eosinófilos em intestino grosso de animais com 90 dias de infecção. O sequenciamento de DNA bacteriano das fezes mostra que houve alteração no microbioma dos animais, porém não há identidade significante, ou seja, acima de 95% na maioria das bactérias. Concluímos que as alterações de histologia da mucosa, a invasão de amastigotas neste tecido e o aumento do LPS, sugerem que a translocação microbiana é um evento ocorrente durante a infecção por L. (L.) infantum neste modelo experimental.
Visceral leishmaniasis, in its active clinical form, is characterized by long-lasting fever, hepatosplenomegaly and cachexia. In Brazil, the lethality is, on average, 7% and the main causes of death are hemorrhage, comorbidity with immunosuppressive diseases and bacterial infection. The mechanism of increased bacterial infection in LV is unclear and one of the hypotheses is that there may be bacterial translocation of the intestinal mucosa to the lumen of the blood vessels and cause a greater severity of the immune-inflammatory response and consequent clinical worsening. The objective of this work was evaluate the occurrence of microbial translocation in Leishmania (L.) infantum infected-hamsters and correlate with the histopathological changes found in the gut of infected animals. Hamsters (Mesocricetus auratus) were infected intraperitoneally with 2x107 amastigotes of L. (L.) infantum and euthanized after 48, 72 hours and 15, 45 and 90 days of infection. As a control group, hamsters were inoculated intraperitoneally with RPMI culture medium. Were collected: blood, feces, spleen, large and small intestines. To detection amastigotes in intestinal mucosa, real-time PCR (qPCR), immunohistochemistry and histopathological analysis were used, and histological alterations in intestinal tissue were also evaluated. Spleen was used to determine the parasitic load through the Stauber technique. To detection of microbial translocation or products related, was performed quantification of lipopolysaccharide (LPS) in plasma. In order to evaluate the possible change in intestinal bacterial flora, bacterial sequencing of sample faeces from control and infected hamsters was carried. We observed increased parasite load on spleen and intestine as the infection progressed, the difference being significant at 90 days of infection. At the same time, we observed increased circulating LPS in infected animals at different times, 48 hours, 72 hours, 45 days and 90 days, with decrease in the intermediate period of 15 days, howeversignificant difference was observed only at 90 days post-infection in relation to control group. Histopathological changes were observed in the large and small intestine, ranging from mild to severe inflammatory infiltrate, enteritis, histiocytosis, and amastigotes. The changes occurred from 48 hours of infection, differentiating the population of the inflammatory infiltrate between neutrophils, lymphocytes, and even eosinophils in the large intestine of animals with 90 days of infection. |Bacterial sequencing shows that there was a change in the microbiome of the animals, but there is no significant identity, ie, above 95% in most bacteria. We conclude that changes in mucosal histology, invasion of amastigotes in this tissue and increase in LPS, suggest that microbial translocation is an event occurring during L. (L.) infantum infection in this experimental model.

Les troubles du spectre de l'autisme (TSA) sont des troubles du neuro-développement caractérisés par des déficits des interactions et de la communication sociales, des comportements répétitifs et intérêts restreints et des anomalies de perception sensorielle. Certains patients TSA ont des troubles gastro-intestinaux (GI), une dysbiose du microbiote intestinal et des niveaux altérés de métabolites produits par ce microbiote. Des travaux supportent l'existence d'un axe microbiote-intestin-cerveau par lequel le microbiote peut communiquer avec le cerveau via des relais périphériques comme le nerf vague, le tractus GI ou encore le système immunitaire. Une voie de communication émergente est via les métabolites microbiens qui peuvent agir sur ces relais périphériques ou directement sur le cerveau. Ainsi, des perturbations de l'axe microbiote-intestin-cerveau pourraient contribuer au développement des symptômes des TSA via les métabolites microbiens.Pendant ma thèse, j'ai étudié le métabolite microbien p-crésol, issu de la dégradation microbienne de la tyrosine alimentaire. Des études cliniques avaient montré que le p-crésol était anormalement élevé chez les patients TSA. J'ai donc exploré l'hypothèse d'un lien de causalité entre niveaux élevés de p-crésol et comportements associés aux TSA. Mes objectifs étaient de préciser les effets du p-crésol sur le comportement et de mieux comprendre les mécanismes périphériques et/ou centraux impliqués.J'ai d'abord contribué à la finalisation d'une étude dans laquelle nous avons montré que des souris adolescentes exposées par voie orale au p-crésol présentaient des déficits du comportement social, des comportements répétitifs, une diminution de l'activité des neurones dopaminergiques impliqués dans le circuit de la récompense sociale, ainsi qu'une dysbiose intestinale associée à la sévérité des troubles comportementaux. J'ai aussi montré que l'exposition au p-crésol altérait la perception des odeurs, mais n'induisait pas d'anxiété, une comorbidité fréquente des TSA. En parallèle, j'ai implémenté un système de suivi longitudinal automatisé du comportement de souris en groupes sociaux et mis en évidence des altérations du comportement social en groupe chez les souris exposées au p-crésol. J'ai par ailleurs pu confirmer l'hypothèse qu'une exposition précoce au p-crésol en période périnatale, affectait le comportement social.Concernant les mécanismes impliqués, j'ai réalisé un phénotypage immunitaire et des explorations fonctionnelles du tractus GI qui m'ont permis d'écarter l'hypothèse d'un effet du p-crésol sur ces deux voies. Ceci a conforté mon hypothèse d'un effet direct du p-crésol au niveau central, et notamment sur la biosynthèse des catécholamines dopamine (DA) et noradrénaline (NA)). Mes résultats suggèrent que le p-crésol administré aux souris par voie orale circule dans le sang et peut atteindre le cerveau. Aussi, j'ai montré que les souris exposées au p-crésol présentaient une diminution des niveaux centraux de DA et NA, ainsi que des activités de la tyrosine hydroxylase et de la DA-β-hydroxylase, enzymes critiques de leur synthèse. De plus, j'ai établi des associations entre les niveaux de p-crésol, les habiletés sociales, la biosynthèse des catécholamines et la composition du microbiote cæcal de ces souris. Enfin, en inhibant pharmacologiquement la DBH, j'ai récapitulé les déficits sociaux présents chez les souris traitées au p-crésol.L'ensemble de ces résultats supportent une action directe du p-crésol sur le cerveau et que la réduction de la biosynthèse des catécholamines pourrait participer aux effets délétères du p-crésol sur le circuit de la récompense et le comportement social. Mes travaux ouvrent la voie à des interventions thérapeutiques ciblant le p-crésol ou les catécholamines pour restaurer les interactions sociales chez les souris exposées au p-crésol. Ceci constituerait une première étape vers de nouveaux traitements des TSA
Autism Spectrum Disorders (ASD) are neurodevelopmental disorders characterized by deficits in social interaction and communication, repetitive behaviours and restricted interests, and abnormal sensory processing. Some ASD patients exhibit gastrointestinal (GI) disorders, intestinal microbiota dysbiosis, and altered levels of metabolites produced by the microbiota. Research supports the existence of a microbiota-gut-brain axis by which the microbiota can communicate with the brain via peripheral relays like the vagus nerve, the GI tract, and the immune system. An emerging route of communication is via microbial metabolites which can act on these peripheral relays or directly on the brain. Though, disruption of the microbiota-gut-brain axis may contribute to the development of ASD symptoms via microbial metabolites.During my thesis, I studied the microbial metabolite p-cresol, derived from the microbial degradation of dietary tyrosine. Clinical studies showed that p-cresol was abnormally elevated in ASD patients. I therefore explored the hypothesis of a causality link between elevated levels of p-cresol and ASD-associated behaviours. My objectives were to identify the effects of p-cresol on behaviour and to better understand the peripheral and/or central mechanisms involved.I first contributed to finalise a study in which we showed that adolescent mice orally exposed to p-cresol exhibited social behaviour deficits, repetitive behaviours, reduced activity of dopamine (DA) neurons involved in the social reward circuit, as well as intestinal dysbiosis associated with the severity of behavioural impairments. I also showed that p-cresol exposure altered odour perception, but did not induce anxiety, a frequent ASD comorbidity. In parallel, I implemented an automated tracking system for the longitudinal study of mouse behaviour in social groups and highlighted that p-cresol-treated mice exhibited social group behaviour deficits. I was also able to confirm the hypothesis that early perinatal exposure to p-cresol altered social behaviour.Regarding the underlying mechanisms, I conducted immune phenotyping and functional explorations of the GI tract, which allowed me to rule out the hypothesis of p-cresol affecting these two pathways. This further supported my hypothesis of a direct effect of p-cresol at the central level, specifically on the biosynthesis of the catecholamines dopamine (DA) and noradrenaline (NA). My findings suggest that orally administered p-cresol in mice circulates in the blood and can reach the brain. Additionally, I demonstrated that mice exposed to p-cresol exhibited decreased central levels of DA and NA, as well as decreased activities of tyrosine hydroxylase and DA-β-hydroxylase, critical enzymes in their synthesis. Furthermore, I established associations between p-cresol levels, social abilities, catecholamine biosynthesis and the caecal microbiota composition of these mice. Finally, by pharmacologically inhibiting DBH, I recapitulated the social deficits present in p-cresol-treated mice.Taken together, these results support a direct action of p-cresol on the brain, and suggest that the reduction in catecholamine biosynthesis may contribute to the deleterious effects of p-cresol on the reward circuit and social behaviour. My work paves the way for therapeutic interventions targeting p-cresol or catecholamines to restore social interactions in mice exposed to p-cresol. This could represent a preliminary step towards new treatments for ASD

Les cellules de la coiffe racinaire et les cellules bordantes et apparentées (AC-DC) libèrent un mucilage principalement composé de glycopolymères, mais également d’ADN extracellulaire. Cette matrice mucilagineuse associée aux AC-DC forme une structure dense, connue sous le nom de piège extracellulaire de racine ou RET, qui entoure l'apex racinaire. Dans cette étude nous avons caractérisé la composition du RET du soja (Glycine max) et du pois (Pisum sativum) par immunomarquages de surface et par chromatographie gazeuse. Les résultats ont montré que les polysaccharides majoritairement présents dans le RET sont les pectines, en majorité des RG-I très ramifiés, et les xyloglucanes. Les zones de l’apex racinaire et d’élongation, et le RET ont une composition différente, ce qui suggère une spécificité des tissus, capable d’assurer des fonctions particulières, notamment dans les interactions entre la racine et les microorganismes. Par la suite, nous avons étudié l’effet du RET du soja et du pois sur le comportement de deux bactéries, Pseudomonas fluorescens et Bacillus subtilis, ainsi que sur les zoospores d’un oomycète, Phytophthora parasitica. Pour ce faire, des tests de confrontation ont été menés, puis chaque microorganisme a été suivi à l'aide d'un logiciel d’imagerie et son déplacement a été caractérisé. Ainsi, les observations au microscope ont notamment révélé que les bactéries sont filtrées par le RET, alors que les zoospores ne le sont pas. Toutefois, lorsque les microorganismes pénètrent le réseau mucilagineux, ils présentent une mobilité très fortement affectée, par rapport à ceux restant à l'extérieur du RET. A l’intérieur du réseau, les vitesses de déplacement sont considérablement réduites, d’un facteur trois pour les bactéries et d’un facteur quatre pour les zoospores, avec des trajectoires très fortement altérées. Ces résultats indiquent que le RET du soja et du pois entrave le déplacement des microorganismes et, par conséquent, leur migration vers la racine. Enfin, nous avons tenté de déconstruire le RET via l’utilisation d’enzymes hydrolytiques et suivi les changements par imagerie et par chromatographie d’exclusion stérique. Les résultats ont révélé que le RET était particulièrement résistant aux différents traitements enzymatiques, ce qui est très probablement dû à la composition et l’organisation des polymères dans le RET
Root cap cells and root associated, cap-derived cells (AC-DC) release a dense mucilage composed mainly of glycopolymers, and extracellular DNA. This mucilaginous matrix associated with the AC-DC forms a complex structure, known as the Root Extracellular Trap (RET), which surrounds the root tip. In this study we characterized the composition of the RET of soybean (Glycine max) and pea (Pisum sativum) by using immunocytochemistry and gas chromatography. The results showed that the polysaccharides predominantly present in the RET are pectins, mainly highly branched RG-I, and xyloglucans. The root elongation and meristematic zones and the RET exhibit different composition, which suggests a specificity of the tissues, able to ensuring specific functions, particularly in the interactions between the root and soil microorganisms. Then, we studied the effect of RET from soybean and pea on the behaviour of two bacteria, Pseudomonas fluorescens and Bacillus subtilis, and on the zoospores of an oomycete, Phytophthora parasitica. To this end, comparison tests were carried out, then microorganisms were tracked using imaging software and their movements were characterized. Microscopic observations revealed that bacteria are seived by the RET, while zoospores are not. However, when the microorganisms penetrate the mucilaginous network, their mobility is greatly affected compared with those remaining outside the RET. Within the RET, the speeds are considerably reduced, by a factor of three for bacteria and a factor of four for zoospores, with very strongly altered trajectories. These results indicate that the RET of soybean and pea hinders the movement of microorganisms and, consequently, their migration towards the root. Finally, we attempted to deconstruct the RET using hydrolytic enzymes (i.e. glycosidases and DNase) and monitored the changes using imaging and steric exclusion chromatography. The data revealed that the RET was particularly resistant to the various enzymatic treatments, which is most likely due to the composition and complex organization of the polymers within the RET

More than 270,000 km of rivers and streams are impaired due to fecal pathogens, creating an economic and public health burden. Fecal indicator organisms such as Escherichia coli are used to determine if surface waters are pathogen impaired, but they fail to identify human health risks, provide source information, or have unique fate and transport processes. Statistical and machine learning models can be used to overcome some of these weaknesses, including identifying ecological mechanisms influencing fecal pollution. In this study, canonical correlation analysis (CCorA) was performed to select parameters for the machine learning model, Maxent, to identify how chemical and microbial parameters can predict E. coli impairment and F+-somatic bacteriophage detections. Models were validated using a bootstrapping cross-validation. Three suites of models were developed; initial models using all parameters, models using parameters identified in CCorA, and optimized models after further sensitivity analysis. Canonical correlation analysis reduced the number of parameters needed to achieve the same degree of accuracy in the initial E. coli model (84.7%), and sensitivity analysis improved accuracy to 86.1%. Bacteriophage model accuracies were 79.2, 70.8, and 69.4% for the initial, CCorA, and optimized models, respectively; this suggests complex ecological interactions of bacteriophages are not captured by CCorA. Results indicate distinct ecological drivers of impairment depending on the fecal indicator organism used. Escherichia coli impairment is driven by increased hardness and microbial activity, whereas bacteriophage detection is inhibited by high levels of coliforms in sediment. Both indicators were influenced by organic pollution and phosphorus limitation.

En recherche et en clinique, les états pathologiques du cerveau ont été caractérisés, et de nouvelles stratégies de traitement évoluent au quotidien. Néanmoins, le traitement de l'une des maladies les plus graves, le glioblastome, est dans de nombreux cas sans succès. Dans ce domaine, la radiothérapie représente la méthode la plus efficace. Cependant, comme les cellules tissulaires normales sont également affectées par les effets des rayonnements que les cellules cancéreuses, la dose prescrite reste fortement limitée par les effets indésirables radiotoxiques. Ainsi, une demande continue d’amélioration de techniques d'irradiation défie les chercheurs et les cliniciens à ce jour. Une nouvelle forme de radiothérapie est en cours de développement, la Microbeam Radiation Therapy (MRT). En MRT, les rayons X, générés par une source de lumière synchrotron, sont collimatés en microfaisceaux de quelques dizaines de microns de large et séparés de quelques centaines de microns. Cette géométrie d'irradiation permet un dépôt de dose très élevée dans les trajets des microfaisceaux (dose pic) tandis que les tranches de tissu situées entre eux ne reçoivent que 5 à 10% de la dose pic (dose vallée). Le principal avantage de cette nouvelle modalité réside dans les effets préférentiels sur la tumeur par rapport aux tissus normaux. Les réponses des vaisseaux tumoraux diffèrent considérablement de celles observées dans des vaisseaux sanguins matures, préservant ainsi les tissus normaux tout en permettant l'ablation des cellules cancéreuses. Dans cette thèse, les effets de la MRT sur le tissu cérébral sain ont été étudiés plus en détail. Premièrement, des cerveau entier de rats sains a été exposé à la MRT (doses vallées de 7 à 25 Gy). Deuxièmement, la MRT a été administrée selon plusieurs ports (jusqu'à 5), focalisées dans le noyau caudé droit (dose vallée de 10 Gy). Ces animaux ont été soumis à des tests comportementaux, à une imagerie par résonance magnétique (IRM) et à une analyse histologique jusqu'à un an après l'exposition. Ces résultats ont été comparés à des animaux non traités ou exposés à une irradiation à faisceau plein (Broad Beam, BB), généré à l'hôpital ou au synchrotron. Par ailleurs, l’efficacité thérapeutique de la MRT avec la géométrie à 5 ports a été évaluée sur des rats porteurs de gliosarcome 9L. Un suivi similaire à l’étude précédente a été effectué. Les résultats ont démontré que les effets à long terme de la MRT sur les tissus cérébraux sains à des doses vallées supérieures à 10 Gy n'étaient pas négligeables. Des effets vasculaires chroniques ont débuté à cette dose, alors qu'une nécrose tissulaire n'a été observée qu'après une exposition de 25 Gy. Des changements de comportement ont été observés par une augmentation de la locomotion et de l'exploration. Cependant, les observations vétérinaires n'ont pas soulevé de préoccupation chez les rats irradiés avec une dose vallée de ≤ 17 Gy ou chez les rats exposés à la configuration multiport. Remarquablement, l'ajout successif d'incidences MRT au protocole standard pour le traitement des tumeurs 9L a augmenté de manière significative et exponentielle la survie des animaux et le contrôle tumoral. En effet, la MRT multiport augmente des doses d’équivalences biologiques d’un facteur d'environ 2.5, un résultat jamais atteint par aucune autre approche radiothérapeutique. La préservation des tissus normaux et l'index thérapeutique exceptionnel font de la MRT multiport une méthode innovante et prometteuse qui est prête pour un transfert vers la clinique
In research and in clinics, pathologic states of the brain have been widely characterized, and new treatment strategies evolve on a daily basis. Conversely, therapy of one of the most severe brain conditions, known as glioblastoma, is in many cases without success. Within the therapeutic range, radiotherapy represents the most efficient method. However, as normal tissue cells are equally affected by radiation effects as cancerous cells, the prescribed dose remains greatly limited by radiotoxic adverse reactions. Thus, a continuous demand of improved irradiation techniques challenges researchers and clinicians to date. A novel form of radiotherapy is being developed, termed Microbeam Radiation Therapy (MRT). In MRT, X-rays are generated by a synchrotron light source and are collimated into an array of parallel microbeams that are a few tens of microns wide and separated by a few hundred microns. This irradiation geometry allows very high dose deposition in the microbeam paths (peak dose) while tissue slices located in-between these paths receive only 5-10% of the peak dose (valley dose). The major benefit of this new modality lies within the preferential effects on tumor than on normal tissues. Tumor vessel responses differ drastically from those observed in mature blood vessels, thus preserving normal tissues while successfully ablating cancerous cells. In this thesis, the effects of MRT on normal brain tissue were further investigated. First, normal rats were exposed to whole-brain MRT (valley doses from 7 to 25 Gy). Second, MRT was delivered through multiple ports (up to 5), focalized in the right caudate nucleus (10 Gy valley dose). These animals were subject to behavioral tests, magnetic resonance imaging (MRI) and histologic analysis until one year post exposure. Results were compared to untreated rats and animals exposed to hospital- or synchrotron-generated broad beam (BB) irradiation. In addition, the multiport MRT geometry was tested on 9L gliosarcoma-bearing rats. The results demonstrated that long-term normal brain tissue effects of MRT at valley doses higher than 10 Gy were not negligible. Chronic vascular effects started off this dose, whereas tissue necrosis was only observed after 25 Gy exposure. MRT-induced behavioral changes were seen in increased locomotion and exploratory drive. However, veterinary observations did not raise concern in rats irradiated with ≤17 Gy MRT valley dose or in rats exposed to the multiport configuration. Remarkably, the successive addition of MRT incidences to the standard protocol for 9L tumor treatment increased significantly and exponentially animal survival and tumor control. Indeed, multiport MRT increases biological equivalent doses by a factor of ~2.5, a result never achieved by any other radiotherapeutical approach. The exceptional normal tissue sparing and the outstanding therapeutic index make multiport MRT a promising innovative method that is primed for clinical translation

Yes
The behavior of a linear copolymer of N-isopropyl acrylamide with pendant vancomycin functionality was compared to an analogous highly branched copolymer with vancomycin functionality at the chain ends. Highly branched poly(N-isopropylacrylamide) modified with vancomycin (HB-PNIPAM-van) was synthesized by functionalization of the HB-PNIPAM, prepared using reversible addition-fragmentation chain transfer polymerization. Linear PNIPAM with pendant vancomycin functionality (L-PNIPAM-van) was synthesized by functionalization of poly(N-isopropyl acrylamide-co-vinyl benzoic acid). HB-PNIPAM-van aggregated S. aureus effectively whereas the L-PNIPAM-van polymer did not. It was found that when the HB-PNIPAM-van was incubated with S. aureus the resultant phase transition provided an increase in the intensity of fluorescence of a solvatochromic dye, nile red, added to the system. In contrast, a significantly lower increase in fluorescence intensity was obtained when L-PNIPAM-van was incubated with S. aureus. These data showed that the degree of desolvation of HB-PNIPAM-van was much greater than the desolvation of the linear version. Using microCalorimetry it was shown that there were no significant differences in the affinities of the polymer ligands for D-Ala-D-Ala and therefore differences in the interactions with bacteria were associated with changes in the probability of access of the polymer bound ligands to the D-Ala-D-Ala dipeptide. The data support the hypothesis that generation of polymer systems that respond to cellular targets, for applications such as cell targeting, detection of pathogens etc., requires the use of branched polymers with ligands situated at the chain ends.
MRC

When exposed to the Fenton oxidation process, approximately 25% of the initial TNT was mineralized and approximately 63% was converted to unknown soluble products. When exposed to Fe(0) under acidic conditions, TNT was completely reduced to unknown products, approximately 20% of which were not soluble. Reduction of TNT by Fe(0) did not occur above pH 3.0. When exposed to sulfide, TNT was reduced to a mixture of 4ADNT, 2ADNT, DA6NT and unknown soluble products. TNT and its reduction products from chemical (iron and sulfide), aquatic plant, and Clostridia systems were not mineralized by either an activated sludge culture or a mixed aerobic culture grown on anthranilic acid. Biological transformation of TNT and sulfide-reduced TNT products, however, did appear to occur in the aerobic microbial culture grown on anthranilic acid.

The microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting development and immune function, and modulating behavior. However, more research is needed to elucidate the potential impact of environmental pollutants on host microbial communities and how microbiomes can modulate the toxicity of contaminants to the host. Through a literature review of 18 studies that assessed the impacts of various anthropogenic chemicals on fish-associated microbiomes, we found that toxicants generally decrease microbial diversity, which could lead to long-term health impacts if chronically stressed, and can increase the host’s susceptibility to disease as well as the chemical resistance of certain microbes. These findings led us to explore the impacts of one of the reviewed contaminants, polycyclic aromatic hydrocarbons (PAHs), typically found in oil. The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and had catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). This study tested the hypotheses that exposure to weathered oil would cause significant shifts in fish gut-associated microbial communities, with taxa known for hydrocarbon degradation increasing in abundance and that foraging behavior would decrease, potentially due to microbial dysbiosis via the gut-brain axis. We characterized the gut microbiome (with 16S rRNA gene sequencing) of a native GoM estuarine species, the sheepshead minnow (Cyprinodon variegatus). Fish were exposed to High Energy Water Accommodated Fractions (HEWAF; tPAH = 80.99 ± 12.5 μg/L) of oil over a 7-day period and whole gastrointestinal tracts were sampled for microbiome analyses. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome did not experience any significant changes in alpha or beta diversity but known hydrocarbon degrading taxa were noticeably present in oil-exposed communities and were absent in controls. We found the order Pseudomonadales, the family Paenibacillaceae, and Pseudomonas pachastrellae to be among these, with Pseudomonadales increasing in abundance. Foraging behavior was not significantly affected by oil exposure. This work highlights the need for further research to elucidate the functional metagenomic responses of the fish gut-microbiome under oil spill conditions.

Yes
Liquid crystal is a new emerging biomaterial. The physical property of liquid crystal plays a role in supporting the adhesion of cells. Nano and microball indentation techniques were applied to determine the elastic modulus or viscoelasticity of the cholesteryl ester liquid crystals in the culture media. Nano-indentation results (108 ± 19.78 kPa, N = 20) agreed well with the microball indentation (110 ± 19.95 kPa, N = 60) for the liquid crystal samples incubated for 24 hours at 37o C, respectively. However, nanoindentation could not measure the modulus of the liquid crystal (LC) incubated more than 24 hours. This is due to the decreased viscosity of the liquid crystal after immersion in the cell culture media for more than 24 hours. Alternatively, microball indentation was used and the elastic modulus of the LC immersed for 48 hours was found to decrease to 55 ± 9.99 kPa (N = 60). The microball indentation indicated that the LC did not creep after 40 seconds of indentation. However, the elastic modulus of the LC was no longer measurable after 72 hours of incubation due to the lost of elasticity. Microball indentation seemed to be a reliable technique in determining the elastic moduli of the cholesteryl ester liquid crystals.
Science Fund Vot. No. S024 or Project No. 02- 01-13-SF0104 and FRGS Vot. No. 1482 awarded by Malaysia Ministry of Education

Day-roosts are an essential resource for tree-hole roosting microbats (Microchiroptera), providing shelter, protection from predators and an appropriate microclimate for energy conservation and reproduction. Microbats often make use of multiple roosting sites, shifting between roosts frequently. Conservation of tree-hole roosting microbats requires an understanding of roost selection and fidelity to enable the protection of sufficient suitable roosting sites. In Australia, as in other countries, habitat loss, particularly in the form of large hollow-bearing trees, is threatening the survival of microbat populations. In addition, the renewal of natural roosts in Australia is very slow, as trees may need to be 100 years old for hollows to form. Where roosting resources are limited, such as in urbanised areas, batboxes may be used as a substitute. As bat-boxes are also accessible to researchers, these roosting sites can help to improve our understanding of roosting behaviour.
This thesis investigates the roosting behaviour of two sympatric microbat species: Gould’s wattled bat (Chalinolobus gouldii) and the white-striped freetail bat (Tadarida australis). These are insectivorous tree-hole roosting species, which naturally occur in urban Melbourne, Australia. Both species make use of bat-boxes at three sites in Melbourne, often sharing roosts with members of the other species. This provided an opportunity not only to study their use of bat-boxes for conservation management purposes, but to investigate factors influencing bat roost selection and fidelity. This study incorporated PIT tags (microchips) and a detector array at the bat-boxes, in addition to monthly manual bat-box inspections, as a method for monitoring roost-use. This approach enabled the collection of long-term, fine-scale roosting data. These data, along with captive and field-based experiments were used to examine the influence of parasites, microclimate and social structure on roost selection patterns and roost fidelity. The specific questions posed were whether tree-hole roosting bats: select roosts based on physical characteristics; perceive a cost of carrying ectoparasites and avoid infested roosts; select roosts to maintain social associations; and select for specific beneficial microclimates.
The patterns of roost selection, ectoparasite diversity, social structure, and the selection of roost microclimate differed between the two species. Microclimate of the bat-boxes was a strong influence on roost selection for both species, as it is for microbats generally. White-striped freetail bats preferred warmer roosts with stable humidity. For Gould’s wattled bats, the selection of roost microclimate differed between the sexes and even between separate, but adjacent, roosting groups. Patterns of preference indicated that individuals had knowledge of the available roosting sites.
The presence of parasites had no obvious influence on roost selection patterns in either species. The white-striped freetail bat was found to support lower ectoparasite diversity, which may be influenced by characteristics of the pelage and may partially explain why parasite load was not a useful predictor of roost selection in this species. In contrast, Gould’s wattled bat supported a larger diversity of ectoparasites, which showed clear patterns of distribution through the bat populations, and intra-specific and spatial variability. A radio-tracking study indicated that parasites in the roost and on the Gould’s wattled bat may influence their roosting behaviour. Additionally, experimental assessments of the bats’ grooming response to parasites indicated that the perceived costs of these parasites differed with parasites that remained permanently attached to the host eliciting a stronger response than those also found in the roost. The defensive mechanism against parasites that completed part of their life-cycle in the roost was expected to be avoidance behaviour, yet, in both captive and field experiments, these parasites did not strongly influence roost selection or fidelity.
Social associations among white-striped freetail bats appeared to be random, and did not explain roosting patterns. This may reflect the restricted sampling of roosting sites, and the possible role of the bat-boxes in this study as ‘satellite’ roosts, separate from a larger communal roost, likely to be in a large tree-hollow. Unlike white-striped freetail bats, Gould’s wattled bats showed fission-fusion social structure, driven by stronger female associations. The distribution and abundance of parasites was correlated with the social structuring of the host species, and host selection appeared to facilitate transmission. These patterns suggest that female Gould’s wattled bats, in particular, are choosing roosts based on the benefits of social association despite the cost of increased parasite risk, and may provide an explanation for sexual segregation in temperate tree-roosting bats.
This study demonstrates the species-specificity of roosting behaviour, and the importance of investigating several factors that influence roost selection, to better understand roost requirements. It also highlights the inherent complexity in roost selection by tree-hole roosting microbats, which may be making trade-offs between the benefits of social associations and the cost of parasitism, as well as choosing an optimal microclimate. Further investigation into interactions between these factors will greatly advance our understanding of roost selection and fidelity in tree-hole roosting bats.