Submitted by Almir Azevedo (barbio1313@gmail.com) on 2017-06-23T12:20:40Z No. of bitstreams: 1 dissertação mestrado ien 2015 Fagner Santos do Carmo.pdf: 2634463 bytes, checksum: 492adfb185de630bd9eb4395153ea9a5 (MD5) / Made available in DSpace on 2017-06-23T12:20:41Z (GMT). No. of bitstreams: 1 dissertação mestrado ien 2015 Fagner Santos do Carmo.pdf: 2634463 bytes, checksum: 492adfb185de630bd9eb4395153ea9a5 (MD5) Previous issue date: 2015-06 / O câncer de próstata é o segundo tipo mais prevalente em homens no mundo inteiro, originando milhares de mortes todos os anos e apresentando elevadas taxas de incidência futura de novos acometimentos. Comparada as técnicas diagnósticas disponíveis, a conjugação de aptâmeros a radionuclídeos, mostra-se eficaz na entrega de drogas a processos tumorais, onde há a capacidade de rastreamento da rota fisiológica percorrida pelo radiomarcado e da mensuração do concentrado em regiões anatômicas específicas através da PET/CT. A afinidade e a especificidade dos aptâmeros promovem o direcionamento seletivo do radioisótopo a tecidos e órgãos MUC1positivos. Dessa forma, este trabalho objetivou a radiomarcação do anti-MUC1 aos radioisótopos 68Ga e 22Na como agente de imagiologia molecular in vivo para o diagnóstico precoce e preciso do processo carcinogênico em próstata. A radiomarcação do aptâmero anti-MUC1 com o 68Ga e o 22Na foi avaliada com a incubação do oligonucleotídeo diretamente a soluções individuais dos radioisótopos em diferentes tempos e temperatura. Apresentando-se eficiente em todos os processos independentemente das variações aplicadas. Corridas cromatográficas em CCD foram aplicadas obtendo marcação de um composto com pureza radioquímica média de 89,07% (89,07±7,79) para o 68Ga e 90,37% (90,37±13,61) para o 22Na, valores superiores ao fator de aceitação para a implementação clínica. Para confrontar os resultados da CCD, a solução radiomarcada também foi sujeita a análises em CLAE, onde foi confirmada a radiomarcação do aptâmero anti-MUC1 com o 68Ga no tempo de retenção médio de 4,842 minutos (4,842±0,03) e 3,679 (3,679±0,51) para o 22Na. No intuito de avaliar o comportamento do oligonucleotídeo radiomarcado in vivo, o anti-MUC1 foi conjugado ao 99mTc e administrado a ratos Wistar sadios, revelando rápido clareamento sanguíneo (0,10±0,06), alta captação do material radioativo no rins (esquerdo 0,94±011 e direito 1,01±0,32) devido a excreção preferencial ser renal. A baixa captação em fígado (0,19±0,09) evidenciou a estabilidade do composto radiomarcado e insignificativas captações de órgãos abdominais mostraram a tendência da minimização dos efeitos da radiação de fundo. Não foi possível detectar a captação cerebral, portanto o complexo não atravessa a barreira hematoencefálica e todos os outros órgãos obtiveram captação em faixa muito baixa ou não detectável. Os resultados de marcação e biodistribuição descritos neste estudo determinaram o potencial do aptâmero anti-MUC1 radiomarcado aos radioisótopos emissores de prósiton 68Ga e 22Na a atuar como agente de imagiologia molecular na área da medicina nuclear.

A malaria humana é uma doença infecciosa transmitida por um mosquito que está atrelada a uma alta taxa de mortalidade. Dentre os parasitas que causam a malaria humana a espécie Plasmodium falciparum é responsável por 90% dos casos letais. Sua virulência é ocasionada por modificações na célula hospedeira provenientes do tráfego de proteínas para a superfície de eritrócitos infectados. Com o intuito de interferir com estas proteínas, aplicamos a técnica Cell- SELEX (do inglês: Systematic Evolution of Ligands by Exponential Enrichment). Após ciclos iterativos de seleção obtivemos moléculas de DNA simples- fita, conhecidas como aptâmeros, que possuem alta afinidade e especificidade contra proteínas alvo presentes na superfície de eritrócitos infectados por P. falciparum. Ensaios de pull- down usando aptâmeros selecionados em sistema de purificação por streptavidina-biotina seguidos por análises de espectrometria de massa revelaram a interação destas moléculas de DNA com proteínas como RIFIN, PfEMP, RAP1, entre outras incorporadas na superfície de eritrócitos infectados. Como consequência destas interações, os aptâmeros selecionados demostraram ser inibidores potenciais na proliferação dos parasitas, como demonstrado por testes in vitro. Outra estratégia usada foi a produção de aptâmeros contra a piridoxal quinase de P. falciparum (PfPdxK) expressa. Usando tanto SELEX-proteína, bem como, ensaios de eletroforese capilar, selecionamos aptâmeros com capacidade para inibir a atividade enzimática da PfPdxK. Nossos dados constituem evidências sobre as aplicações da tecnologia SELEX no desenho racional de compostos contra a malária humana. / The human malaria is a mosquito-borne infectious disease associated with a high risk of mortality. Among the parasite species that causes human malaria, Plasmodium falciparum is responsible by 90% of the lethal cases. The virulence of this pathogen is associated with host cell modifications by trafficking proteins to the surface of infected erythrocytes. Aiming to interfere with these proteins we have applied the Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology. After iterative cycles of selection we obtained single stranded DNA molecules, known as aptamers, with high binding affinity and specificity against protein targets present in the surface of erythrocytes infected with P. falciparum. Pull-down assays using the selected aptamers in a streptavidin-biotin affinity assay followed by mass spectrometry analysis revealed the interaction of these DNA molecules with proteins such as RIFIN, PfEMP, RAP1, among others embedded in the surface of infected erythrocytes. As consequence of these interactions, the selected aptamers demonstrated to be potent inhibitors of parasite proliferation, as validated by in vitro tests. Another strategy used was the production of aptamers against the recombinantly expressed plasmodial pyridoxal kinase (PfPdxK). Using both protein-SELEX as well as capillary electrophoresis assays, we selected aptamers with capacity to inhibit the enzymatic activity of PfPdxK. Our data constitute evidences about the application of SELEX technology in the rationale design of inhibitors against human malaria.

La terminaison de la transcription est essentielle, aussi bien pour assurer la formation de l’extrémité 3’ de transcrits fonctionnels que pour éviter les phénomènes d’interférence transcriptionnelle entre des régions transcrites adjacentes. Ceci est particulièrement important dans un génome compact comme celui de S. cerevisiae. La terminaison est aussi l’une des stratégies principales que la cellule emploie pour contrôler et limiter la transcription dite envahissante ou cachée. Chez S. cerevisiae, l’ARN polymérase II est responsable de la transcription des ARNm et de nombreuses classes d’ARN non codants tels que les sn(o)ARN et les CUT (Cryptic Unstable Transcripts). Ces derniers représentent une fraction importante des transcrits issus de la transcription cachée. Il existe deux voies canoniques de terminaison de la transcription par cette polymérase. Elles font intervenir le complexe de clivage et de polyadénylation, CPF-CF, notamment pour la terminaison des ARNm ou le complexe NNS pour la terminaison des sn(o)ARN et des CUT. Au cours de ma thèse j’ai étudié deux aspects de la terminaison de la transcription : 1) l’étude des motifs de recrutement du complexe NNS et 2) l’identification et la caractérisation d’une nouvelle voie de terminaison par le facteur Rap1. Les complexes CPF-CF et NNS agissent tous les deux en liant le transcrit naissant et l’ARN pol II. Le complexe NNS lie l’ARN naissant grâce à ses sous-unités Nrd1 et Nab3 qui reconnaissent des motifs spécifiques. Cependant, bien que la séquence de ces motifs soit maintenant connue, leur présence ne permet pas de définir de façon certaine un terminateur. En effet, le nombre de ces motifs varie beaucoup d’un terminateur à l’autre. Afin de mieux comprendre la structure des terminateurs ciblés par le complexe NNS et l’organisation des motifs liés par Nrd1 et Nab3, j’ai recherché les séquences impliquées dans la terminaison d’un CUT modèle en réalisant une mutagenèse aléatoire et j’ai identifié par SELEX des motifs de fixation optimale du dimère Nrd1-Nab3. Un second volet de ma thèse porte sur la caractérisation d’une nouvelle voie de terminaison de la transcription dépendante du facteur Rap1. Rap1 est important pour la structure des télomères et c’est aussi un facteur de transcription ciblant des centaines de promoteurs. Il active ou réprime l’initiation de la transcription notamment en recrutant des complexes de remodelage de la chromatine sur les promoteurs ciblés. De façon surprenante, le motif de fixation de ce facteur a été identifié dans des séquences capables de terminer la transcription isolées au laboratoire. Mes travaux ont permis de caractériser le mécanisme de terminaison par Rap1 et de distinguer cette voie des voies de terminaison canoniques. Ce facteur, lié à l’ADN, agit comme une barrière en bloquant la progression de l’ARN polymérase II par un mécanisme de « road-block ». Les polymérases ainsi arrêtées sont ciblées par une voie qui permet leur élimination lorsqu’elles sont bloquées par des dégâts sur l’ADN, impliquant leur ubiquitination et vraisemblablement leur dégradation par le protéasome. Les ARN libérés sont polyadénylés par la poly(A)-polymérase Trf4 et dégradés par l’exosome nucléaire. Ce mécanisme de terminaison est utilisé dans un contexte naturel puisque j’ai identifié des transcrits endogènes de S. cerevisiae terminés par cette voie. Nous proposons que la terminaison par Rap1 contribue au contrôle de la transcription envahissante. Ce facteur assurerait ainsi au niveau des promoteurs qu’il lie une double fonction de facteur de transcription et de protection de ces promoteurs contre l’interférence transcriptionnelle. / Transcription termination is essential, both for the 3’ end formation of functional transcripts and to avoid transcriptional interference between adjacent transcription units. This is particularly important in a compact genome such as S. cerevisiae. Termination is also one of the main strategies used by the cell to control and limit the “pervasive” or “hidden” transcription. In S. cerevisiae, RNA pol II is responsible for the transcription of the mRNAs and numerous non-coding RNA families such as the sn(o)RNAs and the CUTs (Cryptic Unstable Transcripts). CUTs represent a large fraction of the “pervasive” or “hidden” transcription. There are two canonical transcription termination pathways for this RNA polymerase. They involve the cleavage and polyadenylation complex (CPF-CF), in particular for the mRNAs termination, or the NNS complex for sn(o)RNAs and CUTs termination. During my thesis I studied two aspects or the transcription termination: 1) the motifs involved in the NNS complex recruitment on RNA and 2) the identification and the characterization of a new termination pathway by Rap1. CPF-CF and NNS complex are both recruited on the nascent transcript and on the RNA pol II. The NNS complex binds the RNA through its subunits Nrd1 and Nab3 which recognize specific motifs. Nonetheless, even if these motif sequences are now known, their presence does not elicit the certain identification of NNS dependent terminators. To clarify the NNS dependent terminator structure and the organization of the motifs bound by Nrd1 and Nab3 I looked for the sequences involved in a specific CUT termination doing a random mutagenesis experiment and I identified by SELEX the Nrd1-Nab3 dimer optimal binding motifs. A second part of my thesis concerns the characterization of a new transcription termination pathway dependent on the Rap1 factor. Rap1 is important for the telomere structure and it is also a transcription factor that targets hundred of promoters. It activates or represses transcription initiation recruiting chromatin remodeling complexes on the targeted promoters. Surprisingly, the Rap1 binding motifs have been identified among sequences eliciting termination isolated in the laboratory. My work has led to the characterization of the termination mechanism by Rap1 and distinguished this pathway from the two canonical pathways. This factor, bound to DNA, acts as a barrier blocking the RNA pol II progression by a road-block mechanism. These arrested polymerases are targeted by a pathway responsible for the elimination of RNA pol II blocked by DNA damages, implying their ubiquitination and probably their degradation by the proteasome. The released RNAs are polyadenylated by the poly(A) polymerase Trf4 and degraded by the nuclear exosome. This termination mechanism is used in a natural context since I identified S. cerevisiae endogenous transcripts terminated by this pathway. We propose that the Rap1 termination contributes to the pervasive transcription control. This factor could elicit, on its bound promoters, a double function of both transcription factor and protection of these promoters against transcriptional interference.

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.

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 β3-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 β3-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.

L’objectif de ma thèse a été de développer de nouvelles sondes utilisables en imagerie biomédicale, basées sur l’utilisation d’aptamères, pour la détection de deux types de tumeurs : les glioblastomes et les mélanomes. Les traceurs développés ont pour objectif de cibler la protéine matricielle hMMP-9. Lors d’une thèse précédente, un aptamère anti hMMP-9 noté F3B a été obtenu. Afin de transformer cet aptamère en une sonde pour l’imagerie biomédicale, différents conjugués F3B ont été préparés: Cy5, DOTA ou MAG3. L’affinité des nouveaux conjugués pour l’hMMP-9 a été évaluée par SPR et sur coupes de tumeurs. Des études de biodistribution des conjugués F3B-DOTA et F3B-MAG3 ont été réalisées sur des souris portant le mélanome, les résultats ont montré que les deux aptamères marqués détectent spécifiquement l’hMMP-9. De plus, la détection de la protéine hMMP-9 par le conjugué F3B-CY5 a été confirmée par imagerie de fluorescence. Afin d’améliorer la sensibilité de détection des tumeurs, deux types de modifications ont été envisagées, développer des structures multimériques de F3B et élaborer d’un système bi-fonctionnel. Pour ces deux approches, nous avons synthétisé un dendrimère à point focal, pouvant donner accès à une imagerie multi-modale. Ce dendrimère porte deux ou trois bras espaceur porteurs d’un groupement azoture utilisé pour le couplage avec les aptamères par la chimie «click». Le dendrimère porte au point focal une fonction amine NH2 utilisée pour fixer une biotine, afin de déterminer l'affinité Kd de cette nouvelle sonde par SPR. Par la suite un ligand DOTA sera fixé afin de pouvoir visualiser ce traceur en TEMP. / The aim of my thesis was to develop new probes that can be used in biomedical imaging, based on the use of aptamers for the detection of two types of tumors: glioblastomas and melanomas. Tracers have been developed with the aim to target the matrix protein hMMP-9. In a thesis, an aptamer anti-hMMP-9 noted F3B was obtained. Based on this compound, different derivatives were prepared for using in biomedical imaging: F3B-Cy5, F3B-DOTA or F3B-MAG3. The affinity of the new conjugates for hMMP-9 was evaluated by SPR and on sections of human melanomas. Biodistribution studies of F3B-DOTA conjugates and F3B-MAG3 were performed at on melanoma bearing mice, the results showed that both radiolabeled aptamers specifically detected the hMMP-9. In addition, optical fluorescence imaging confirmed the binding to hMMP-9 by F3B-CY5. In order to improve tumor detection sensitivity, two types of modifications were investigated: developing of F3B multimeric structures and a bi-functional system. For both these approaches, we synthesized a dendrimer with a focal point, which could give access to a multi-modal imaging. This dendrimer has two or three spacers bearing an azide group used for coupling with aptamers by "click" chemistry. The dendrimer carrying at the focal point an amine function NH2 used for fixing biotin in order to determine the affinity Kd of this new probe by using SPR. A DOTA ligand will be fixed later in order to view this tracer in SPECT.

La détection de petites molécules est d'un grand intérêt dans les domaines pharmaceutique, environnemental, alimentaire et de la biologie clinique. Les aptamères, sélectionnés par la méthode SELEX (pour Systematic Evolution of Ligands by Exponential Enrichment), sont des oligonucléotides qui se lient à une cible donnée avec une affinité et une spécificité importantes. L'objectif de ce travail est d'établir de nouvelles méthodologies analytiques basées sur l'utilisation des aptamères pour la détection de petites molécules. Dans un premier temps, une méthodologie par électrophorèse capillaire, dérivée du concept de déplacement du brin complémentaire de l'aptamère, est décrite pour la détection simultanée de plusieurs analytes dans un seul capillaire. La deuxième étude se focalise sur le développement d'un aptacapteur colorimétrique simple, rapide et peu coûteux, qui utilise le concept général de protection enzymatique de l'aptamère et les nanoparticules d'or en tant que système de transduction. Enfin, deux méthodes par polarisation de fluorescence, basées sur le concept de déplacement (du brin complémentaire ou de l'aptamère lui-même), sont présentées afin d'accroitre les potentialités des aptacapteurs dédiés à la détection des petites molécules. / Small biomolecule detection is of great interest and importance in the pharmaceutical, environmental, food and clinical fields. Aptamers, selected by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), are oligonucleotides that bind to a target with high affinity and specificity. The objective of the work is to establish novel methodologies of aptamer-based assays for the small biomolecule detection. In the first work, a rationalized capillary electrophoresis strategy, derived from the structure-switching aptamer concept, is described for the design of simultaneous detection of multiple analytes. The second work based on a gold nanoparticle colorimetric sensing strategy allows a rapid, label-free, homogeneous assay for small molecule using an aptamer enzymatic cleavage protection strategy. In the third work, two aptamer-based fluorescence polarization approaches, using the displacement concept, are described to improve the potentialities of the small molecule-dedicated aptasensors.

Les séquences riches en guanines, qu’elles soient ADN ou ARN, peuvent former des structures non canoniques à quatre brins appelées quadruplexes de guanines ou G-quadruplexes. Ces structures reposent sur la formation et l’empilement de quartets de guanines ; elles peuvent être trouvées dans de nombreuses régions du génome. Des motifs G-quadruplexes apparaissent fréquemment lors de la sélection d'aptamères par SELEX : on constate un biais dans la proportion de guanines par rapport aux autres nucléotides dans les bases de données regroupant les séquences d'aptamères connus. Nous avons entrepris une analyse systématique, in silico puis in vitro, de motifs aptamères décrits dans la littérature. Nous avons utilisé un algorithme de prédiction actuellement développé au sein du laboratoire dans le but de déterminer quelles sont les séquences susceptibles de former des G-quadruplexes in silico. Afin de vérifier ces prédictions, un test biophysique permettant de cribler rapidement de nombreuses séquences a été mis en place. Nos résultats démontrent que de nombreux aptamères publiés sont susceptibles de se replier en G-quadruplexe. Un autre volet de ce travail concernait la mise en pratique du SELEX avec deux objectifs distincts. Nous avons d'abord effectué une sélection in vitro contre un ligand de G-quadruplexes, afin de préciser quels motifs nucléiques peuvent interagir avec cette molécule. Nous avons réalisé ensuite des expérience de SELEX contre plusieurs G-quadruplexes ADN ou ARN biologiquement pertinents (motif télomérique humain, répétitions minisatellites et séquences présentes dans les UTR de différents ARNm), dans le but d'obtenir des sondes spécifiques de ces conformations. / Guanine-rich DNA or RNA sequences can adopt non-canonical structures composed of four strands called guanines quadruplexes or G-quadruplexes. These structures are made by the formation and stacking of guanine quartets; they can be found in various region of the genome. G-quadruplexes motifs are frequently found during the selection of aptamers by the so-called SELEX method: a bias exists in the proportion of guanines in comparison with other nucleotides in a database gathering together known aptamers sequences. We did an in silico then in vitro systematic analysis of aptameric motifs described in the literature. We used a prediction algorithm currently developed in the laboratory to determine which sequences car form G-quadruplexes in silico. In order to check those predictions, a biophysical test allowing to quickly assay a lot of sequences was set up. Our results demonstrate that a lot of published aptamers are able to fold into G-quadruplexes. Another part of this work is related to the use of the SELEX with two different goals. First of all we did an in vitro selection against a G-quadruplexes ligand, in order to tell exactly which nucleic motifs can interact with this molecule. We then carried out SELEX experiments against several DNA or RNA biologically relevant G-quadruplexes (human telomeric motifs, minisatellite repetitions and sequences from UTR of mRNA), with the aim of getting specific probes for these conformations.