Ngan, Kit Shan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 176-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

Pour aider à la protection de l’environnement et au diagnostic de maladies, il est nécessaire de disposer de méthodes d’analyse performantes. Ces méthodes doivent être rapides et peu coûteuses afin d’analyser un grand nombre d’échantillons et détecter la présence éventuelle de la molécule recherchée. C’est dans ce cadre qu’un bio-essai à double reconnaissance dédié aux petites molécules a été développé. Il repose sur l’utilisation d’aptamères comme élément de reconnaissance moléculaire et sur la formation de « kissing complex ». Les aptamères sont des oligonucléotides qui possèdent une grande spécificité et une grande affinité pour leur cible. Les « kissing complexes » désignent, quant à eux, l’interaction de deux séquences au niveau de leur boucle apicale. L’objectif de la thèse est de continuer l’étude et l’optimisation de ce schéma d’analyse, puis montrer sa potentialité et sa versatilité. Dans un 1er temps, la détection en multiplexe de plusieurs petites molécules a été mise en place. L’optimisation du système de détection a ensuite été effectuée pour la détection de l’adénosine avec le développement d’une nouvelle stratégie de détection. Enfin, un test colorimétrique basé sur l’utilisation de nanoparticules d’or a été décrit. / Bioanalytical tools based on molecular recognition elements constitute one of the most important strategies used to routinely quantify low molecular weight analytes in biological fluids or environmental matrices as disease-related biomarkers, drugs or toxins/contaminants. Recently, a novel aptamer-based sensing design has been reported. It is based on a dual recognition mechanism that involves the formation of functional nucleic acid architectures, named “kissing complex”. Aptamers are single stranded oligonucleotides that possess high affinity and high selectivity for their target. The aim of this thesis is to pursue the study and the optimization of this sandwich like scheme and to prove its potential. Firstly, the simultaneous detection of multiple small analytes by fluorescence anisotropy using the sandwich like scheme has been reported. Then, this novel bioassay has been optimized through the development of a new strategy. Finally, a gold nanoparticle colorimetric

Les aptamères correspondent à de courtes séquences d'oligonucléotides possédant une forte affinité et spécificité envers un ligand (petites molécules organiques, peptides, acides nucléiques, protéines, cellules). Du fait de leurs remarquables propriétés, ils sont utilisés comme alternative aux anticorps dans les dispositifs de type biocapteur/biopuce, notamment pour la détection de petites molécules (PM < 2000 Da). L'imagerie de résonance des plasmons de surface (SPRi) est une technique de détection optique qui a gagné une attention croissante ces dernières années. Elle est basée sur un principe de variation de l'indice de réfraction d'une surface sélective lors de l'interaction sonde/cible. Sa sensibilité est néanmoins limitée aux molécules de poids moléculaire supérieur à 2000 Da. Dans le cadre de ces travaux, nous avons développé une biopuce à aptamères pour à la détection d'une petite molécule, l'adénosine, au moyen de la technique de résonance des plasmons de surface (SPR). Pour cela, deux différentes stratégies ont été développées. La première combine l'utilisation de nanoparticules d'or (AuNPs) pour l'amplification du signal SPRi avec l'ingénierie des séquences d'aptamères. La seconde stratégie est basée sur l'exploitation de la stabilité thermodynamique apportée par l'interaction de la cible (adénosine) avec les séquences d'aptamères. Le dispositif SPR est alors couplé à un système de régulation de température, permettant ainsi d'assurer la dissociation des complexes et d'établir des profils de dénaturation caractéristiques. Nos résultats initient ainsi une nouvelle approche dans la détection de petites molécules par SPRi et ouvrent de nouvelles perspectives de développement des biocapteurs à aptamères. / Aptamers are single-stranded DNA (ssDNA) or RNA molecules capable of binding to target molecules, including proteins, metal ions and drugs. Because of their specific binding abilities and many advantages over antibodies (higher stability, lower cost, easy chemical modification…), they provide a great opportunity to produce sensing surfaces for effective and selective detection of small molecules. Surface Plasmon Resonance imaging (SPRi) has become one of the most widely used label-free method for the study of biorecognition events on sensor surfaces. This technique provides a rapid approach, however, limited by low refractive index changes occurring when small molecules (<2000 Da) are captured on the sensor. Whereas significant reflectivity variation is observed upon the interaction of large molecules like proteins with the sensing interface, for small molecules targets such adenosine, the reflectivity variation is often too small to be detected by SPRi. Thereby, only few studies have been reported so far on SPRi-based biosensor for small molecules detection using aptamers. In this work, we developed two bioassay strategies for the detection of a model small molecule, adenosine, using Surface Plasmon Resonance imaging. The first one combines the SPRi signal enhancement effect induced by gold nanoparticles (AuNPs) with the advantage of using engineered DNA aptamers. The experimental results have demonstrated that the presence of gold nanoparticles and adenosine, which works as a molecular linker between engineered aptamer fragments, can significantly increase the SPRi response. The second strategy is based on the thermodynamics of binding between adenosine and its aptamer. To that end, SPRi technique was coupled with rigorous temperature control and aptamer duplex stability was monitored (affected by target binding) by quantification of melting transitions. Our results initiate a new approach for small molecule detection using SPRi with the aim to validate future prospects for integration in parallelized platform.

Le SELEX est une technique d’évolution moléculaire dirigée qui permet, après plusieurs tours de sélection, d’enrichir une banque d’acides nucléiques en séquences capable de se lier de manière spécifique à une cible. Le séquençage est utilisé pour identifier ces séquences que l’on nomme « aptamères ». Depuis l’arrivée récente du séquençage à haut débit (HD), il est possible d’analyser des millions de séquences. L’objectif de la thèse était de développer des méthodes pour traiter et analyser les données de séquençage HD afin de faciliter l’identification des meilleurs aptamères d’un SELEX. Au cours de cette thèse, un test robotisé de liaison sur cellules adhérentes vivantes a été mis au point pour mesurer l’affinité d’aptamères issus de SELEX ciblant des cellules (cell-SELEX). Puis, l’évolution de l’abondance des séquences d’un cell-SELEX a été analysée par séquençage HD. Ceci nous a permis de concevoir une nouvelle approche phylogénétique baptisée FREDROGRAM. Cette approche évolutive a permis d’identifier des mutants avec une meilleure affinité au sein d’une famille d’aptamères issu de ce cell-SELEX. Enfin, le séquençage HD de deux SELEX dirigés contre des protéines a contribué à mieux comprendre l’impact des paramètres de sélection sur la population de séquences et à identifier de nouveaux aptamères, notamment en réduisant le nombre de tours de SELEX. En conclusion, ces travaux montrent l’utilité du séquençage HD pour l’identification des meilleurs aptamères et suggèrent de nouvelles pratiques pour la conduite des SELEX futurs. / SELEX is a directed molecular evolution technic which allows, after several rounds of selection, enriching a library from random nucleic acids to sequences able to bind specifically a target. Sequencing technics are then used to identify these sequences called « aptamers ». Since the arrival of High-Throughput Sequencing (HTS), it is now possible to analyse millions of sequences. The aim of the thesis was to develop methods for the treatment and the analysis of HTS data, in order to facilitate the identification of the best aptamers inside a SELEX. During this thesis, a semi-automatic binding test on adherent living cells has been developed to measure the affinity of aptamers identified in SELEX directed against specific cells (cell-SELEX). Then, the evolution of the sequence enrichment during a cell-SELEX has been analysed by HTS. This analysis gave us the possibility to design a new phylogenetic approch named FREDROGRAM. This evolutive approch allowed to identify variants of an aptamer’s family with a better affinity. Finally, HTS of two SELEX directed against proteins has contributed to a better understanding of the impact of selection parameters on the library and to identified new aptamers, notably by reducing the number of SELEX rounds. To conclude, this work shows the importance of HTS in the identification of the best aptamers and suggests new protocols to monitor the next SELEX in a different manner.

Les ARN (Acide RiboNucléique) remplissent de nombreuses fonctions clés dans le vivant. Ils peuvent être support de l'information génétique, régulateurs de celle-ci. Visualiser ces molécules au sein d'une cellule représenterait une étape importante vers une meilleure compréhension de la régulation de l'expression des gènes. Les ARN fluorogènes tels que Spinach et Mango sont des outils extrêmement prometteurs pour atteindre cet objectif. Cependant ces deux ARN fluorogènes présentent une brillance limitée. La Compartimentation in vitro assistée par microfluidique (µCIV) est un outil très prometteur dont notre groupe a démontré l’efficacité pour l’évolution d’ARN. Dans le cadre de cette thèse, la µCIV a été adaptée à la sélection d'aptamères d'ARN fluorogènes pour en améliorer les propriétés (surtout la brillance). De plus, l’utilisation conjointe du séquençage haut débit a permis l’optimisation très rapide et semi-automatisée à la fois d’aptamères mais aussi de biosenseurs fluorogènes. Ainsi, cette thèse a permis de mettre en place et d’exploiter des technologies de criblage robustes pour la découverte de nouveaux aptamères d'ARN et de biosenseurs. / RNA is a key molecule in gene expression and its regulation. Therefore, being able to monitor RNA through live-cell imaging would represent an important step toward a better understanding of gene expression regulation. RNA-based fluorogenic modules are extremely promising tools to reach this goal. To this end, two light-up RNA aptamers (Spinach and Mango) display attractive properties but they suffer from a limited brightness. Since previous work in the group demonstrated the possibility to evolve RNA using microfluidic-assisted in vitro compartmentalization (µIVC), this technology appeared to be well suited to improve light-up aptamers properties by an evolution strategy. Therefore, the µIVC procedure was adapted to fluorogenic RNA aptamers to improve their properties (especially the brightness). Finally, using µIVC in tandem with high-throughput sequencing (NGS) allowed further developing the technology into a more integrated and semi-automatized approach in which RNAs and biosensors are selected by µIVC screening and the best variants identified by a bioinformatics process upon NGS analysis. To summarize, this thesis allowed establishing robust µIVC screening workflows for the discovery of novel efficient light-up RNA aptamers as well as metabolites biosensors.

Foi demonstrado que o gosto doce é transduzido por receptores acoplados a proteína G classe III (GPCRs), T1R2 e T1R3. Essas proteínas exibem longas extremidades amino-terminais que formam um domínio de ligação globular extracelular. Elas são expressas em células associadas ao gosto (células epiteliais que constituem os botões gustativos nas papilas gustativas), que respondem a moléculas associadas ao gosto doce. Quando T1R2 e T1R3 são co-expressas em células heterólogas, elas respondem, como heterômeros, a uma série de açúcares, alguns D-aminoácidos, edulcorantes artificiais e proteínas doces. Foi também demonstrado que o receptor humano T1R2/T1R3 para o gosto doce apresenta múltiplos sítios de ligação. Para melhor compreender a estrutura desse receptor e responder à pergunta de como um único quimiorreceptor pode ser responsivo a uma variedade de ligantes, foi utilizada a abordagem denominada evolução sistemática de ligantes por enriquecimento exponencial (SELEX) para isolar, a partir de uma biblioteca combinatória de oligonucleotídeos, aptâmeros de RNA resistentes a nuclease que se ligam ao receptor humano para o gosto doce com alta afinidade. Após um enriquecimento de doze ciclos do pool original de RNA contendo em torno de 1013 sequências diferentes (contra preparações de membrana de células HEK293T que expressam hT1R2/hT1R3) e outros ciclos de contrasseleção negativa (para eliminar moléculas de RNA que se ligam de forma inespecífica à membrana de nitrocelulose e a outras proteínas diferentes do alvo, ou seja, proteínas de membrana de células HEK293T selvagem), realizou-se a transcrição reversa do RNA seguida de amplificação por PCR e sequenciamento. Aptâmeros do ciclo 12 com sequências consenso foram selecionados, e a ligação de alguns deles com hT1R2/hT1R3 foi então avaliada. Cinco desses aptâmeros mostram claramente uma maior afinidade por células HEK293T que expressam hT1R2/hT1R3. Como segunda parte desta tese, estudamos outro receptor, denominado CD36, que, como o receptor T1R2/T1R3, é expresso na língua. Estudos indicam que ele age como receptor gustativo de gordura. Neste trabalho, verificamos que essa proteína é expressa em uma subpopulação de neurônios olfatórios presentes no epitélio olfatório, indicando que ela pode ter também uma função olfatória, ainda não caracterizada. / It has been shown that sweet taste is transduced by the Class III G Protein-Coupled Receptors (GPCRs) T1R2 and T1R3, which show long N-termini that form a globular extracellular ligand-binding domain. These receptors are expressed in the taste cells (epithelial cells that constitute the taste buds in taste papillae) that respond to sweet tastants, and when T1R2 and T1R3 are coexpressed in heterologous cells, they respond, as heteromers, to a series of sugars, some D-amino acids, artificial sweeteners and sweet proteins. It has also been demonstrated that the sweet taste receptor has multiple binding sites. In order to better understand the structure of this receptor and answer the question of how a single chemoreceptor can respond to a variety of ligands, we used the combinatorial oligonucleotide library screening approach, denominated Systematic Evolution of Ligands by Exponential Enrichment (SELEX), to isolate nuclease-resistant RNA aptamers that bind to the human sweet taste receptor with high affinity. Following a twelve round enrichment of the previous random RNA pool containing around 1013 different sequences (against membrane preparations of hT1R2/hT1R3-expressing HEK293T cells) and negative counterselection cycles (to eliminate RNA molecules that bind nonspecifically to the nitrocellulose membrane and to proteins other than the target, that is, HEK293T cells membrane proteins), the RNA was reverse-transcribed for DNA sequencing. Aptamers from cycle 12 with consensus sequences were selected, and the binding of some of them to the human sweet taste receptor was then evaluated. Five out of the aptamers clearly show greater affinity for hT1R2/hT1R3-expressing HEK293T cells than for hT1R2/hT1R3-non-expressing HEK293T cells. In this thesis we have also analyzed another receptor, denominated CD36, which is also expressed in the tongue. Studies indicate that it acts as a receptor for fat. In this work, we found that CD36 is expressed in a subset of the olfactory neurons localized in the olfactory epithelium, indicating that it may also have an as yet uncharacterized olfactory function.

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como &#946;3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal. / Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as &#946;3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies.

Foi demonstrado que o gosto doce é transduzido por receptores acoplados a proteína G classe III (GPCRs), T1R2 e T1R3. Essas proteínas exibem longas extremidades amino-terminais que formam um domínio de ligação globular extracelular. Elas são expressas em células associadas ao gosto (células epiteliais que constituem os botões gustativos nas papilas gustativas), que respondem a moléculas associadas ao gosto doce. Quando T1R2 e T1R3 são co-expressas em células heterólogas, elas respondem, como heterômeros, a uma série de açúcares, alguns D-aminoácidos, edulcorantes artificiais e proteínas doces. Foi também demonstrado que o receptor humano T1R2/T1R3 para o gosto doce apresenta múltiplos sítios de ligação. Para melhor compreender a estrutura desse receptor e responder à pergunta de como um único quimiorreceptor pode ser responsivo a uma variedade de ligantes, foi utilizada a abordagem denominada evolução sistemática de ligantes por enriquecimento exponencial (SELEX) para isolar, a partir de uma biblioteca combinatória de oligonucleotídeos, aptâmeros de RNA resistentes a nuclease que se ligam ao receptor humano para o gosto doce com alta afinidade. Após um enriquecimento de doze ciclos do pool original de RNA contendo em torno de 1013 sequências diferentes (contra preparações de membrana de células HEK293T que expressam hT1R2/hT1R3) e outros ciclos de contrasseleção negativa (para eliminar moléculas de RNA que se ligam de forma inespecífica à membrana de nitrocelulose e a outras proteínas diferentes do alvo, ou seja, proteínas de membrana de células HEK293T selvagem), realizou-se a transcrição reversa do RNA seguida de amplificação por PCR e sequenciamento. Aptâmeros do ciclo 12 com sequências consenso foram selecionados, e a ligação de alguns deles com hT1R2/hT1R3 foi então avaliada. Cinco desses aptâmeros mostram claramente uma maior afinidade por células HEK293T que expressam hT1R2/hT1R3. Como segunda parte desta tese, estudamos outro receptor, denominado CD36, que, como o receptor T1R2/T1R3, é expresso na língua. Estudos indicam que ele age como receptor gustativo de gordura. Neste trabalho, verificamos que essa proteína é expressa em uma subpopulação de neurônios olfatórios presentes no epitélio olfatório, indicando que ela pode ter também uma função olfatória, ainda não caracterizada. / It has been shown that sweet taste is transduced by the Class III G Protein-Coupled Receptors (GPCRs) T1R2 and T1R3, which show long N-termini that form a globular extracellular ligand-binding domain. These receptors are expressed in the taste cells (epithelial cells that constitute the taste buds in taste papillae) that respond to sweet tastants, and when T1R2 and T1R3 are coexpressed in heterologous cells, they respond, as heteromers, to a series of sugars, some D-amino acids, artificial sweeteners and sweet proteins. It has also been demonstrated that the sweet taste receptor has multiple binding sites. In order to better understand the structure of this receptor and answer the question of how a single chemoreceptor can respond to a variety of ligands, we used the combinatorial oligonucleotide library screening approach, denominated Systematic Evolution of Ligands by Exponential Enrichment (SELEX), to isolate nuclease-resistant RNA aptamers that bind to the human sweet taste receptor with high affinity. Following a twelve round enrichment of the previous random RNA pool containing around 1013 different sequences (against membrane preparations of hT1R2/hT1R3-expressing HEK293T cells) and negative counterselection cycles (to eliminate RNA molecules that bind nonspecifically to the nitrocellulose membrane and to proteins other than the target, that is, HEK293T cells membrane proteins), the RNA was reverse-transcribed for DNA sequencing. Aptamers from cycle 12 with consensus sequences were selected, and the binding of some of them to the human sweet taste receptor was then evaluated. Five out of the aptamers clearly show greater affinity for hT1R2/hT1R3-expressing HEK293T cells than for hT1R2/hT1R3-non-expressing HEK293T cells. In this thesis we have also analyzed another receptor, denominated CD36, which is also expressed in the tongue. Studies indicate that it acts as a receptor for fat. In this work, we found that CD36 is expressed in a subset of the olfactory neurons localized in the olfactory epithelium, indicating that it may also have an as yet uncharacterized olfactory function.

Orientador: Luiz Claudio Nogueira Mendes / Coorientador: Sergio Moraes Aoki / Banca: Flavia Lombardi Lopes / Banca:Gisele Zoccal Mingotti / Banca:Maria denise Lopes / Banca: Eduardo Harry Birgel Junior / Resumo: Novas metodologias para a sexagem de sêmen são buscadas devido à citometria de fluxo apresentar limitações e desvantagens e os aptâmeros surgem como potenciais candidatos para a identificação de espermatozoides conforme a presença do cromossomo X ou Y. Os objetivos foram avaliar o uso de três aptâmeros desenvolvidos para se ligarem especificamente ao espermatozoide Y na separação magnética por sexo de espermatozoides de touro, e desenvolver uma técnica de qPCR que permita realizar facilmente o diagnóstico da proporção sexual em amostras de sêmen. Protocolos de separação por magnetismo utilizando aptâmeros biotinilados foram utilizados em sêmen comercial, e duas frações de espermatozoides foram obtidas para cada aptâmero testado (livre e retida). A proporção relativa de espermatozoides Y foi analisada em reações de qPCR desenvolvidas para este objetivo. A curva padrão utilizada na qPCR foi eficiente para a quantificação da proporção sexual relativa em amostras de sêmen convencional e sexado. Diferença significativa da proporção relativa de Y foi encontrada na fração de espermatozoides livres do aptâmero C12 em relação ao controle (47,7% vs. 51,3%, respectivamente; P = 0,009); as demais separações não apresentaram diferença significativa. Os aptâmeros selecionados não produziram o efeito desejado de separação do sêmen conforme a presença do cromossomo X ou Y, nestas condições utilizadas, mas a diminuição da proporção relativa de Y em uma das frações não retidas indica ser possí... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sex preselection of livestock offspring represents a big potential for genetic improvement. There are a search for new methods to sex-sort bull semen in order to avoid the limitations of flow cytometry/cell sorting technique. Aptamers emerge as potential candidates for sperm identification relative the presence of chromosome X or Y. In this work, the objectives were 1, to evaluate three aptamers developed to specifically binding to the Y-bearing sperm in magnetic separation by sex of bovine sperm and 2, to develop a SYBR Green Real-Time PCR method to determinate sex ratio in bovine semen in semen samples. Separation protocols by magnetism using biotinylated aptamers were used in commercial semen, and two fractions of sperm were obtained for each tested aptamer (free and trapped). The relative ratio of Y-bearing spermatozoa was analyzed in qPCR reactions developed for this purpose. The standard curve for qPCR was efficiently designed to quantify the sex relative ratio of sorted and unsorted semen samples. Significant difference in the relative proportion of Y-bearing sperm was found in the fraction of free sperm samples from aptamer C12 compared to the control samples (47.7% vs. 51.3%, respectively; P = 0.009); other separations showed no significant difference. Selected aptamers did not produce the wanted effect of sperm separation by the presence of chromosome X or Y, in these conditions used, but decreased relative proportion of Y-bearing sperm in one of the free fractions ... (Complete abstract click electronic access below) / Doutor

La maladie de Parkinson (PD) est considérée comme la deuxième maladie neurodégénérative la plus fréquente. L'examen post-mortem de patients parkinsoniens et des modèles physiologiques d’études de la maladie de Parkinson suggèrent la participation de la mort cellulaire programmée, l'inflammation et l'autophagie dues au stress oxydatif, à des mutations ou l’agrégation de protéines au sein des neurones DA. Les aptamères peptidiques sont de petites protéines combinatoires, consistitués d’une plateforme (dans notre cas, la thiorédoxine humaine, hTRX) et une boucle variable insérée dans le domaine actif de hTRX. Deux aptamères peptidiques ont été identifiés par la sélection fonctionnelle. L’aptamère peptide 32 (Apta-32) ,est spécifique liant deux paralogues T32 impliqués dans le processus d'endocytose. L’aptamère peptidique 34(Apta-34) lie à une cible "T34", une protéine pro-apoptotique ayant un rôle dans la voie apoptotique provenant du noyau. Le travail de cette thèse visait à étudier la fonction anti-apoptotique de nos deux aptamères peptidiques dans deux modèles d’étude de la maladie de Parkinson: un modèle cellulaire (in vitro) et un modèle transgénique D. melanogaster (in vivo). Deux toxines majeures ont été appliquées dans ce travail, 6-hydroxindopamine (6-OHDA) et le paraquat, un pesticide couramment utilisé. Nos observations montrent que la drosophile exprimant Apta-32 dans tous les neurones ont montré une meilleure résistance après 48h de traitement avec le paraquat comparé à deux autre aptamères peptidiques, Apta-34 et Apta-TRX (sans boucle de contrôle variable). Une autre étude a révélé un défaut dans la phagocytose des corps apoptotiques au cours du développement embryonnaire de la drosophile exprimant Apta-32 dans les macrophages, ce qui suggère qu’Apta-32 pourrait participer à et peut-être interférer avec le processus de l’autophygie, et que Apta-32 pourrait protéger contre l'autophagie induite par paraquat dans les neurones. / Parkinson’s disease is considered as the second most common neurodegenerative disease. Although the cause of the progressive cell loss of PD remains unclear to date, programmed cell death, inflammation and autophagy due to oxidative stress, gene mutations or protein aggregations within DA neuron have been suggested as potential causes. Peptide aptamers are small combinatorial proteins, with a variable loop inserted into a scaffold protein, human thioredoxin, hTRX. They are used to facilitate dissection of signaling networks by modulating specific protein interactions and functions. Two peptide aptamers were identified by functional selection which inhibit Bax-dependent cell death in mammalian models. One peptide aptamer (Apta-32) is binding two paralogues involved in endocytotic trafficking T32. The second peptide aptamer (Apta-34) is binding to a target "T34", a pro-apoptotic protein mediating apoptosis emanating from the nucleus. The work of my PhD thesis aimed to investigate the anti-apoptotic function of our two peptide aptamers in different PD models including cell model (in vitro), brain tissue slice and D. melanogaster (in vivo) ; in particular their impact on neuron survival after exposure to specific toxins. Two major toxins were applied in this work, 6-hydroxindopamine (6-OHDA) and Paraquat, a commonly used pesticide. Our observations indicated that Drosophila expressing Apta-32 in all neurons showed more resistance 48h after treatment with Paraquat, compared to drosophila expressing Apta-34 or TRX. Another study revealed a defect in phagocytosis of apoptotic bodies in drosophila embryo’s expressing Apta-32 in macrophage, suggesting Apta-32 could be involved in, and perhaps interfere with, the process of autophagy. This suggests that Apta-32 could protect against paraquat induced autophagy in neurons.