Orientador: Prof. Dr. Guilherme Cunha Ribeiro / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Evolução e Diversidade, 2017. / O gênero Aphrophila (Diptera, Limoniidae) incluía 19 espécies descritas, 11 das quais estão distribuídas ao da Sul da América do Sul (Argentina e Chile) e 8 na Nova Zelândia (parte do atual continente da Zelândia). Entretanto, o conhecimento sobre a taxonomia e aspectos da biologia evolutiva e biogeografia do grupo é muito limitado. Nesse contexto, foi realizada uma revisão taxonômica e um estudo filogenético do gênero. Foram redescritas e ilustradas todas as espécies previamente descritas além de 15 novas espécies. Uma nova espécie foi descrita para a Ilha Campbell (NZ), 3 para a Argentina e 11 para o Chile. Com base no resultado da análise filogenética, uma classificação foi proposta para o gênero incluindo 5 subgêneros. Visando encontrar o padrão biogeográfico informado pela análise filogenética, foi feita uma análise de sub-árvores livres de paralogia que resultou no seguinte areagrama: ((Ilha Campbell + América do Sul) + Nova Zelândia). Além do estudo do gênero Aphrophila, esta dissertação inclui um estudo no qual uma nova técnica é proposta visando a integração de dados morfológicos e moleculares em Arthropoda. A técnica baseia-se na dissolução de estruturas proteicas em uma solução de proteinase k, que, ao mesmo tempo em que permite a extração de DNA, resulta no clareamento (diafanização). Do ponto de vista do estudo morfológico, o resultado da diafanização é superior ao que é alcançado através da utilização de KOH (método tradicionalmente utilizado na diafanização de insetos). / The genus Aphrophila (Diptera: Limoniidae) included 19 described species, 11 of which are distributed in the Southern South America (Argentina and Chile) and 8 in New Zealand (part of new continent Zealand). However, the knowledge about taxonomy, biology and biogeography of the group is very limited. In this context, a taxonomic revision and a phylogenetic study of the genus were fulfilled. All previously described species, as well as 15 new species, were redescribed and illustrated. For Campbell Island (NZ) are described one new species, for Argentina 3 new species and for Chile 11 new species. Based on the result of phylogenetic analysis, a classification was proposed for the genus including 5 subgenera. Aiming to find the biogeographic pattern informed by phylogenetic analysis, an analysis of paralogy-free subtrees was made that resulted in the following areagram: ((Campbell Is. + South America) + New Zealand). In addition to the study of the genus Aphrophila, this dissertation includes a study in which a new technique is proposed aiming the integration of morphological and molecular data in Arthropoda. The technique relies on the dissolution of protein structures in a solution of proteinase k, which results in clarification (diaphanization) while allowing DNA extraction. From the morphological point of view, the result of the diaphanization is superior to what is achieved through the use of KOH (method traditionally used in the diaphanization of insects).

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Les biocapteurs sont des moyens d’analyse en plein essor à la fois rapides, sélectifs et peu coûteux applicables à des domaines extrêmement variés (environnement, santé, agroalimentaire,…). Dans ce type d’outil, un élément sensible de nature biologique (anticorps, enzyme, microorganisme, ADN…) doté d’un pouvoir de reconnaissance pour un analyte ou un groupe d’analytes est associé à un transducteur pouvant être de type électrochimique, optique ou thermique. Dans ce travail, nous nous sommes intéressés au développement de différents biocapteurs se basant sur l'immobilisation d'enzymes ou de bactéries sur des microélectrodes en vue d’une détection électrochimique. Nous avons montré les potentialités d’application de deux biocapteurs conductimétriques à base de protéinase K ou de protéinase K et de pronase à la détection des modifications de conformation de la myoglobine et de l’albumine de sérum bovin au cours de leur relargage à partir de microsphères de poly (ε-caprolactone). Nous avons également mis au point un biocapteur conductimétrique à base de catalase et d’alcool oxydase pour une détection rapide et sensible des alcools ainsi que deux biocapteurs à catalase pour la détection impédimétrique et conductimétrique du cyanure et l’étude des interactions catalase-cyanure. Nous avons enfin élaboré des biocapteurs bactériens à base de Pseudomonas putida F1 pour la détection du trichloroéthylène dans les eaux souterraines. Pour cela, une voie originale d’immobilisation des cellules, basée sur la fonctionnalisation du transducteur à l’aide de couches autoassemblées et d’anticorps, ainsi que l’utilisation de nanotubes de carbone, a été explorée / The development of biosensors is an expanding research area. Indeed, biosensors are rapid, selective and cost-effective analytical tools which find applications in various fields (environment, health, food,…). They are constituted of a sensitive biological element (antibody, enzyme, microorganism, DNA…), which can selectively recognize one analyte or a group of analytes, associated to an electrochemical, optical or thermal transducer. In this work, we developed different biosensors based on enzymes or bacteria immobilised onto microelectrodes in view of electrochemical detection. First, we demonstrated the potentialities of two conductometric biosensors based on proteinase K or proteinase K and pronase for the detection of myoglobin and bovine serum albumine conformation changes during their release from poly (ε-caprolactone) microspheres. Then, we elaborated a bi-enzymatic conductometric biosensor with catalase and alcohol oxidase as sensing elements, for a rapid and sensitive detection of alcohols. Catalase impedimetric and conductometric biosensors were also developed for cyanide detection and used for the study of catalase-cyanide interactions. Finally, we prepared Pseudomonas putida F1 whole cell biosensors for the determination of trichloroethylene in groundwaters. For that, an original route, including the functionalisation of the transducer with a self-assembled-monolayer and antibodies, and the use of single-wall carbon nanotubes, was investigated for cell immobilisation

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...

Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...

La caractérisation de nanomatériaux dans des matrices alimentaires et animales suscite un intérêt scientifique important afin d’évaluer les risques potentiels de l’exposition liés à l’utilisation grandissante des nanomatériaux par plusieurs industries, y compris un certain nombre d’applications agroalimentaires. Un facteur limitant à l’étude et la réglementation des nanomatériaux dans des matrices complexes telle que la nourriture est l’absence de méthodes standardisées pour l’extraction et l’analyse de nanoparticules, tout en évitant l’altération de certaines caractéristiques physicochimiques des nanoparticules. Les travaux présentés dans ce mémoire abordent l’optimisation de plusieurs approches de préparation d’échantillon (hydrolyse enzymatique et alcaline) pour l’extraction de nanoparticules d’Ag préalablement équilibrées dans une matrice de boeuf haché mi-maigre. Les nanoparticules extraites ont été analysées par spectrométrie de masse à plasma à couplage inductif en mode particule unique (SP-ICP-MS) permettant la mesure de leur taille et concentration, mais aussi de la concentration en métal dissous, le tout à de très faibles concentrations (de l’ordre du ng/L). La validation de l’analyse par SP-ICP-MS a été réalisée par évaluation de la répétabilité, de la détermination des limites de détection et par une investigation de l’influence du traitement de données sur l’interprétation des résultats. Les pertes de nanoparticules lors de la préparation des échantillons ont été minimisées par l’identification et l’optimisation de paramètres clés tels que la composition du médium d’extraction, l’utilisation d’ultrasons et de la manipulation de l’échantillon après dégradation de la matrice. Les meilleurs recouvrements ont été obtenus par hydrolyse alcaline de la matrice en utilisant de l’hydroxyde de tetramethylammonium (TMAH), mais les échantillons obtenus étaient moins stables et plus susceptibles aux altérations des propriétés physicochimiques des nanoparticules que pour la dégradation par hydrolyse enzymatique utilisant lipase et pancréatine de porc. / The regulation and characterization of nanomaterials in foods and animal matrices are of great interest due to the potential risks associated with their exposure and the increasing number of instances where they are used within the food industry. One factor limiting the scientifically rigorous regulation of nanoparticles in foods is the lack of standardized procedures for the extraction of nanoparticles (NP) from complex matrices, without alteration of their physico-chemical properties. To this end, two sample preparation approaches (enzymatic- and alkaline-based hydrolyses) were tested and optimized in order to extract 40 nm Ag NP, following their equilibration with a fatty ground beef matrix. Extracted NP were characterized using single particle inductively coupled plasma mass spectrometry (SP-ICP-MS), allowing the determination of NP size and concentrations and also dissolved metal concentrations at trace levels. Validation of the SP-ICP-MS analysis was achieved by an evaluation of the repeatability and accuracy and by a determination of the various detection limits. Finally, we also looked into the influence of data treatment on interpretation of the results. NP losses during the sample preparation were minimized by identifying and optimizing key parameters such as the composition of the extraction media, usage of ultrasonication or the handling of the sample after separation from the undigested matter, among other points. The alkaline approach using TMAH (tetramethylammonium hydroxide) was found to have the highest recoveries, however processed samples were found to be less stable and more prone to alteration of the Ag NP physicochemical characteristics than samples processed using an enzymatic digestion based upon pork pancreatin and lipase.

Proteins and their intricate network of interactions are fundamental to many molecular processes that govern life. Mass spectrometry-based structural proteomics represents a powerful set of techniques for characterizing protein structures and interactions. The last decade has witnessed a large-scale adoption in the application of these techniques toward solving a variety of biological questions. Addressing these questions has often been coincident with the further development of these techniques. Insight into the structures of individual proteins and their interactions with other proteins in a proteome-wide context has been made possible by recent developments in the relatively new field of chemical crosslinking combined with mass spectrometry. In these experiments crosslinking reagents are used to capture protein-protein interactions by forming covalent linkages between proximal amino acid residues. The crosslinked proteins are then enzymatically digested into peptides, and the covalently-coupled crosslinked peptides are identified by mass spectrometry. These identified crosslinked peptides thus provide evidence of interacting regions within or between proteins. In this dissertation the development of tools and methods that facilitate this powerful technique are described. The primary arc of this work follows the development and application of mass spectrometry-based approaches for the identification of protein crosslinks ranging from those which exist endogenously to those which are introduced synthetically. Firstly, the development of a novel strategy for comprehensive determination of naturally occurring protein crosslinks in the form of disulfide bonds is described. Secondly, the application of crosslinking reagents to create synthetic crosslinks in proteins coupled with molecular dynamics simulations is explored in order to structurally characterize the intrinsically disordered tau protein. Thirdly, improvements to a crosslinking-mass spectrometry method for defining a protein-protein interactome in a complex sample is developed. Altogether, these described approaches represent a toolset to allow researchers to access information about protein structure and interactions. / Graduate