Taxon composition and biodiversity analyses are known powerful parameters for environmental site status and environment diagnosis. Many ecological studies assess taxon composition through traditional species identification and use bioindicator species to evaluate environmental conditions. The recent breakthrough in bulk sample sequencing combined with DNA barcoding has created a new era for environmental monitoring. Metabarcoding approaches are more robust in studying alpha, and beta diversity compare to the DNA barcoding and the conventional method of species identification, particularly for rare and cryptic species. Here we built upon ecological studies of bioindicator species and transferred the traditionally named taxa to DNA-based approaches. We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques. To evaluate our approach, we have also studied the importance of database’s size and the depth of sequencing in taxonomic classification of high-throughput DNA sequences. / Thesis / Master of Science (MSc) / We developed a small customized DNA database for biodiversity assessment and taxonomic identification of environmental DNA samples using high-throughput amplicon sequences. It contains macroinvertebrate species that are known as indicators of specific environmental conditions. By implementing this small database into the KRAKEN algorithm for the first time, we were able to assess environmental biodiversity compared to other popular methods of taxonomic classification, especially in polluted environments where the taxonomic composition globally change by the presence of anthropogenic drivers. Our method is incredibly faster, and it requires significantly less computational power in contrast to common homology-based techniques.

DNA nanotechnology has been developed in order to construct nanostructures and nanomachines by virtue of the programmable self-assembly properties of DNA molecules. Although DNA nanotechnology initially focused on spatial arrangement of DNA strands, new horizons have been explored owing to the development of the toehold-mediated strand-displacement reaction, conferring new dynamic properties to previously static and rigid structures. A large variety of DNA reconfigurable nanostructures, stepped and autonomous nanomachines and circuits have been operated using the strand-displacement reaction. Biological systems rely on information processing to guide their behaviour and functions. Molecular computation is a branch of DNA nanotechnology that aims to construct and operate programmable computing devices made out of DNA that could interact in a biological context. Similar to conventional computers, the computational processes involved are based on Boolean logic, a propositional language that describes statements as being true or false while connecting them with logic operators. Numerous logic gates and circuits have been built with DNA that demonstrate information processing at the molecular level. However, development of new systems is called for in order to perform new tasks of higher computational complexity and enhanced reliability. The contribution of secondary structure to the vulnerability of a toehold-sequestered device to undesired triggering of inputs was examined, giving new approaches for minimizing leakage of DNA devices. This device was then integrated as a logic component in a DNA-based computer with a retrievable memory, thus implementing two essential biological functions in one synthetic device. Additionally, G-quadruplex logic gates were developed that can be switched between two topological states in a logic fashion. Their individual responses were detected simultaneously, establishing a new approach for parallel biological computing. A new AND-NOT logic circuit based on the seesaw mechanism was constructed that, in combination with the already existing AND and OR gates, form a now complete basis set that could perform any Boolean computation. This work introduces a new mode of kinetic control over the operation of such DNA circuits. Finally, the first example of a transmembrane logic gate being operated at the single-molecule level is described. This could be used as a potential platform for biosensing.

No description available.

A great effort has been recently devoted to the development of new devices for the detection of specific sequences of DNA, due to increasing need of label - free, fast, cheap, and miniaturized analytical systems able to detect target sequences for screening purposes, especially in food industry for genetically modified organisms (GMOs). In this study, development of a QCM - based DNA biosensor for the detection of the hybridisation of CaMV 35S promoter sequence (P35S) was investigated. Attention was focused on the choice of the coating chemistry that could be used for the immobilisation of probe sequences on the gold surface of the quartz crystal. Two immobilisation procedures were tested and compared considering the amount of the immobilised probe, the extent of the hybridisation reaction, the possibility of regeneration and the absence of non - specific adsorption. The two coating methods were based on the use of self - assembled monolayers. One of them employed the interaction between the thiol and gold for the immobilisation of a thiolated P35S probe, while the other employed formation of functionalised aldehyde groups by ethylenediamine plasma polymerization on the gold surface for the immobilisation of amined P35S probes through gluteraldehyde activation. Results indicated that immobilisation of a thiolated probe provides better immobilisation characteristic, higher sensitivity for the detection of the hybridisation reaction, absence of non - specific adsorption and a higher stability with respect to the regeneration step. The optimised immobilisation procedure for the thiolated probe was used for the detection of P35S sequence in PCR - amplified DNAs and in real samples of pflp - gene inserted tobacco plants that produce ferrodoxin like protein additionally. Fragmentation of the genomic DNAs were achieved by digestion with restriction endonucleases and sonication. The obtained results from the fragmented genomic DNAs demonstrated that it is possible to detect the target sequence directly in non-amplified genomic DNAs by using the developed QCM - based DNA biosensor system. The developed QCM-based DNA biosensor represented promising results for a real-time, label - free, direct detection of DNA samples for the screening of GMOs.

Cette thèse porte sur la mise en place et le développement d’une approche expérimentale pour l’étude de la dynamique spatio-temporelle de réseaux de réactions à base d’ADN. Nos résultats démontrent la capacité des réseaux d’ADN à se spatialiser sous la forme d’ondes progressives. Nous avons également pu obtenir des motifs stationnaires à base d’ADN et d’assemblages de billes. Ce travail contribue donc à la conception de motifs spatio-temporels de réactions chimiques et de matériaux par le biais de réseaux réactionnels biochimiques programmables. Nous apportons également de nouvelles données sur l’émergence d’ordre spatio-temporel à partir de processus de réaction-diffusion. De ce fait, cette étude contribue à une meilleure compréhension des principes fondamentaux qui régissent l’apparition d’une auto-organisation moléculaire dans un système chimique hors-équilibre. De plus, la combinaison de réseaux synthétiques d’ADN, du contrôle du coefficient de diffusion de plusieurs espèces d’ADN et de la micro-fluidique peut donner lieu à des motifs spatiaux stables, comme par exemple, les fameuses structures de Turing, ce qui tend à confirmer le rôle de celles-ci dans la morphogénèse. / This PhD work is devoted to developing an experimental framework to investigate chemical spatiotemporal organization through mechanisms that could be at play during pattern formation in development. We introduce new tools to increase the versatility of DNA-based networks as pattern-forming systems. The emergence of organization in living systems is a longstanding fundamental question in biology. The two most influential ideas in developmental biology used to explain chemical pattern formation are Wolpert's positional information and Turing's reaction-diffusion self-organization. In the case of positional information, the pattern emerges from a pre-existing morphogen gradient across space that provides positional values as in a coordinate system. Whereas, the Turing mechanism relies on self-organization by driving a system of an initially homogeneous distribution of chemicals into an inhomogeneous pattern of concentration by a process that involves solely reaction and diffusion. Although numerical simulations and mathematical analysis corroborate the incredible potential of reaction-diffusion mechanisms to generate patterns, their experimental implementation is not trivial. And despite of the exceptional achievements in pattern formation with Belousov–Zhabotinsky systems, these are difficult to engineer, thus limiting their experimental implementation to few available mechanisms. In order to engineer reaction-diffusion systems that display spatiotemporal dynamics the following three key elements must be controlled: (i) the topology of the network (how reactions are linked to each other, i.e. in a positive or negative feedback manner), (ii) the reaction rates and (iii) the diffusion coefficients. Recently, using nucleic acids as a substrate to make programmable dynamic chemical systems together with the lessons from synthetic biology and DNA nanotechnology has appeared as an attractive approach due to the simplicity to control reaction rates and network topology by the sequence. Our experimental framework is based on the PEN-DNA toolbox, which involves DNA hybridization and enzymatic reactions that can be maintained out of equilibrium in a closed system for long periods of time. The programmability and biocompatibility of the PEN-DNA toolbox open new perspectives for the engineering of the reaction-diffusion chemical synthesis, in particular in two directions. Firstly, to study biologically-inspired pattern-forming mechanisms in simplified, yet relevant, experimental conditions. Secondly to build new materials that would self-build by a process inspired from embryo morphogenesis. We worked towards the goal of meeting the two requirements of Turing patterning, transferring chemical spatiotemporal behavior into material patterns, and imposing boundary conditions to spatiotemporal patterns. Therefore, the structure of this document is divided into four specific objectives resulting in four chapters. In chapter 1 we worked on testing a DNA-based reaction network with an inhibitor-activator topology. In chapter 2 we focused on developing a strategy to tune the diffusion coefficient of activator DNA strands. In chapter 3 we explored how chemical patterns determine the shape of a material. Finally, in chapter 4 we addressed the issue of controlling the geometry over a DNA-based reaction-diffusion system. Overall, we have expanded the number of available tools to study chemical and material pattern formation and advance towards Turing patterns with DNA.

No description available.

No description available.

Les chloroacétanilides sont une famille d'herbicides largement utilisée en agriculture, et contribuent de ce fait à la pollution environnementale. Leur devenir, y compris dans les écosystèmes rédox-dynamiques récepteurs comme les zones humides, est encore mal compris. Dans ce travail, la dégradation microbienne de chloroacétanilides (métolachlore, acétochlore et l'alachlore) a été étudiée par des approches innovantes de chimie analytique et de biologie moléculaire, à l'échelle du laboratoire en utilisant des microcosmes en colonnes, et in situ dans des zones humides construites à ciel ouvert et conçues pour traiter les intrants chimiques issus de l'agriculture.Une nouvelle méthode d’analyse isotopique composés-spécifiques a été développée. Les résultats indiquent la biodégradation des chloroacétanilides dans les zones humides, également suggérée par la détection des produits de dégradation correspondants (acides éthane sulfonique et oxanilique). Dans les expériences en microcosme de laboratoire, les chloroacétanilides ont principalement été dégradés dans les zones anoxiques de la rhizosphère, suggèrant un rôle prépondérant des processus anaérobies. L'analyse par chromatographie chirale du métolachlore a en outre révélé la dégradation préférentielle de l'énantiomère S du métolachlore, confirmant l'importance des processus biologiques dans la dissipation des chloroacétanilides. Les corrélations qui ont pu être observées entre les changements de variables hydrochimiques et de conditions hydrauliques et des différences de composition bactérienne détectées par génotypage par polymorphisme de longueur des fragments de restriction (T-RFLP) et par pyroséquençage du gène ARNr 16S confirme le potentiel de bio-indicateurs basés sur l'ADN pour suivre le fonctionnement des écosystèmes.Sur la base de ce travail, la détection et l'identification des micro-organismes et des voies biochimiques responsables de la dégradation de chloroacétanilides dans les zones humides, ainsi que l'élaboration d'indicateurs génétiques bactériens pour le suivi de la dégradation de chloroacétanilides en zones humides, émergent comme autant d’objectifs de recherche à court-terme. / Chloroacetanilide herbicides are widely used in agriculture, and thereby contribute to environmental pollution. Their fate, including in redox-dynamic receptor ecosystems such as wetlands, remains poorly understood. In this work, microbial degradation of chloroacetanilides (metolachlor, acetochlor and alachlor) was investigated by emerging chemical and molecular biological approaches, at the lab-scale using microcosm columns, and in situ, in outdoor constructed wetlands designed for the treatment of chemical pollutants originating from agriculture.A novel compound-specific isotope analysis (CSIA) method was developed, and the results indicated biodegradation of chloroacetanilides in wetlands, which was also suggested by detection of ethane sulfonic acid and oxanilic acid degradation products. In lab-scale wetland microcosms, chloroacetanilides were mainly degraded in anoxic rhizosphere zones, suggesting a predominant role of anaerobic processes. Chiral chromatographic analysis of metolachlor revealed preferential degradation of the (S) enantiomer of metolachlor, and further confirmed the role of biological processes in chloroacetanilide dissipation. Changes in hydrochemical variables and hydraulic conditions correlated with differences in wetland bacterial composition detected by terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing analyses of the bacterial 16S rRNA gene, confirming the potential of DNA-based bioindicators for follow-up of ecosystem functioning.On the basis of this work, detecting and identifying the microorganisms and biochemical pathways responsible for chloroacetanilide degradation in wetlands, as well as developing bacterial gene-based indicators of wetland functioning, emerge as research objectives for the near future.

Qualità e sicurezza degli alimenti, inclusa la loro tracciabilità ed autenticità, è diventato ngli ultimi anni obiettivo primario per la salute e il benessere dei consumatori. Il progetto è diretto allo sviluppo e applicazione di metodiche DNA-based per la tracciabilità di specie vegetali, varietà di frumento duro e microorganismi a difesa della qualita’, salubrita’ ed autenticità della filiera grano e prodotti processati. Le attività progettuali dello studio sono finanziate da industria (Barilla S.P.A.) con il coinvolgimento di enti pubblici di ricerca (Università Cattolica Del Sacro Cuore Di Piacenza, CREA-GB di Fiorenzuola D’arda). Il progetto si articola in tre argomenti principali: WP1.Tracciabilità di specie vegetali nella filiera pasta. Tracciabilità di varietà di frumento duro nella filiera pasta A questo scopo sono state intraprese nel secondo anno di attività due azioni dirette allo sviluppo e validazione di due diverse metodiche di fingerprinting varietale. Partendo dalle direttive UPOV in materia di impiego di marcatori molecolari per la caratterizzazione varietale è stata applicata e validata su di un pool di 26 varietà di interesse per l’industria un’ analisi basata su di una combinazione di marcatori SSR (Simple Sequence Repeats) che ha consentito di identificare in maniera univoca ciascuna delle varietà in esame. Il saggio è stato trasferito ai laboratori dell’industria che lo applica attualmente nella routine per il controllo di partite di granella. A fronte della robustezza dell’analisi SSR si pongono però i lunghi tempi analitici. Per ottimizzare questo aspetto si è completata un’attività di sequenziamento parziale del genoma di 28 varietà presenti in due repliche biologiche (52 campioni) e 12 mix di DNA di due varietà (4 differenti percentuali per ogni coppia di varietà miscelate). I dati ottenuti hanno fornito circa 15.000 marcatori molecolari DArT-seq (Diversity Array Technology ) e SNP (Simple Nucleotide Polimorphisms). Dall’intero set di marcatori è stato quindi individuato, attraverso una procedura bioinformatica, un set ridotto di marcatori ad alta informatività in grado di identificare univocamente le singole varietà e di predire la presenza di altre varietà in miscela e la percentuale di contaminazione. WP2.Tracciabilità Di Microrganismi Fungini Nella Filiera Pasta Questo studio è volto al controllo e identificazione di specie microbiche patogene che possono svilupparsi lungo la filiera grano con conseguente impatto negativo sulla salubrità di granella, di semole e dei prodotti finiti. A questo scopo sono stati prodotti campioni di granella a contaminazione controllata. Si è costituita attraverso l’analisi delle sequenze Barcode una ceppoteca che comprende i maggiori patogeni fungini che possono contaminare la granella durante la crescita della pianta in campo o durante lo stoccaggio della granella. Dopo l’inoculo artificiale dei singoli ceppi in due varietà di frumento duro, sono stati raccolti, a tempi crescenti, campioni di spighe e granella. La metodica è risultata rapida e sensibile nell’identificazione di DNA fungino fin dalle prime fasi dell’infezione, quando i sintomi della malattia risultavano ancora non ancora visibili. Le informazioni di sequenza Barcoding, in prospettiva, potranno essere utilizzate per sviluppare nuovi metodi di identificazione fungina più sensibili e rapidi. WP3. Identificazione molecolare di microrganismi vivi o morti in pesto L’obiettivo di questo lavoro è stato quello di sviluppare metodiche di V-qPCR (Viability-PCR), per permettere l’identificazione, quantificazione e discriminazione cellule microbiche vive o morte in alimentiprocessati, come il pesto. Per lo studio è stato scelto il batterio patogeno B.cereus, microrganismo ubiquitario, patogeno e sporigeno di difficile identificazione soprattutto in matrici complesse. Durante questo lavoro è stato sviluppato un protocollo analitico che prevede l’estrazione del DNA batterico da pesto, matrice interferente e complessa. Parte del lavoro è stata svolta presso l’Istituto di Microbiologia dell’Università Cattolica e l’Istituto IATA CSIC Institut d’Agroquímica i Tecnologia dels Aliments in Valencia. / Food quality and safety, including food traceability and authenticity, have become crucial in the last decades. Today, molecular and genetic progress can support the agri-food industry, due to the improvement of new analytical tools. Among the available applications, DNA-based methods can detect the presence of a particular species or variety along the food supply chain, verify the genetic identity of food and feed ingredients and detecting the presence of contaminating organisms, thus becoming an essential tool to study patterns, causes, and risk factors of diseases and outbreaks. As a consequence, genetic analysis has become increasingly popular even among non-specialists and highly beneficial for consumers, agricultural farmers, governments, and the private sector (Reid, O’Donnell, and Downey 2006). In this framework, the research developed in this thesis arises by active collaboration between the private company Barilla G. & R. Fratelli S.p.A., the public research institute CREA-GB (Consiglio per la Ricerca in agricoltura e l'analisi dell Economia Agraria) and Università Cattolica del Sacro Cuore, to develop a set of DNA-based methods to improve the traceability and authenticity of plant and microbial species and durum wheat varieties applicable from farm to fork. Following these aims, the research developed in this thesis includes: 1. The optimization and validation of qPCR assay for the discrimination of plant species along the pasta production chain through the organization of a ring test involving nine Italian public and private laboratories. The results obtained in this study were published in the Journal of Cereal Science (Chapter 2); 2. The discrimination of durum wheat varieties by selecting SSRs and DarT molecular markers as reliable methods for variety fingerprinting (Chapter 3). The results confirm the sensitivity of the method and the feasibility to 7 protect the food industry from fraud and ensure the consumer a certified pasta quality; 3. The application of the Barcoding technique and the development of qPCR assay for the identification and quantification of field fungi (Fusarium, Alternaria, Michrodochium, Cochliobolus spp.) and saprophytic fungi (Aspergillus, Penicillium, Rhizopus spp) along the wheat chain (Chapter 3). The sensitivity of the method was investigated by inoculating potted durum wheat plants at full anthesis and wheat kernels (pre and postharvest trials). The DNA-based methods demonstrate a key role in pathogen detection and the application in several points of the wheat chain (e.g., for control of both locally and imported grains, for storage lots, to evaluate the environmental risk associated with grain powder for farmers and workers); 4. The optimization of Viability q-PCR (V-qPCR) for the discrimination of dead and alive Bacillus cereus, a spore-forming bacteria (Chapter 4). The results of PMAxx, combined with qPCR, have demonstrated the selective discrimination of B.cereus viable cells, with no false-positive signals determined by dead cells, a peculiar aspect of thermally treated food; 5. The comparison of two DNA extraction kits (FastDNA® SPIN Kit for Soil – MB and NucleoSpin Tissue - Macherey Nagel) by detecting B.cereus spores in basil pesto sauce, selected as a model food matrix. Despite the limit of detection (LOD) achieved (respectively 1.8x102 spores/gr by using Fast DNA TM SPIN and 2.7 x 105 spores/gr by using NucleoSpin®), the principal challenge remains the spores' DNA extraction from the complex matrix. Lastly, the results obtained during the doctoral research project were globally discussed (Chapter 5).

Prior to European settlement, black bear (Ursus americanus) were far less abundant in the state of California. Estimates from statewide harvest data indicate the California black bear population has tripled in the last 3 decades. Bears inhabit areas they formally never occurred (e.g., urban environments) and populations that were at historically low densities are now at high densities. Though harvest data are useful and widely used as an index for black bear population size and population demographics statewide, it lacks the ability to produce precise estimates of abundance and density at local scales or account for the numerous bears living in non-hunted areas. As the human population continues to expand into wildlife habitat, we are being forced to confront controversial issues about wildlife management and conservation. Habituated bears living in non-hunted, urban areas have been and continue to be a major concern for wildlife managers and the general public. My objective was to develop DNA-based capture-mark-recapture (CMR) survey techniques in wildland and urban environments in Mono County, California to acquire population size and density at local scales from 2010 to 2012. I also compared population density between the urban and wildland environment. To my knowledge, DNA-based CMR surveys for bears have only been implemented in wildland or rural environments. I made numerous modifications to the techniques used during wildland DNA-based CMR surveys to survey bears in an urban environment. I used a higher density of hair-snares than typically used in wildland studies, non-consumable lures, modified hair-snares for public safety, included the public throughout the entire process, and surveyed in the urban-wildland interface as well as the city center. These methods were efficient and accurate while maintaining human safety. I determined that there is likely a difference in population density between the urban and wildland environments. Population density was 1.6 to 2.5 times higher in the urban study area compared to the wildland study area. Considering the negative impacts urban environments can have on wildland bear populations, this is a serious management concern. The densities I found were similar to those found in other urban and wildland black bear populations. The baseline data acquired from this study can be used as part of a long-term monitoring effort. By surveying additional years, population vital rates such as apparent survival, recruitment, movement, and finite rate of population change can be estimated.