This study embarks on exploiting the Thickness Shear Mode (TSM) acoustic wave sensor and the ElectroMagnetic Piezoelectric Acoustic Sensor (EMPAS) towards the study of aptamer-to-cocaine binding in a label-free direct approach. The high sensitivity and selectivity offered by the EMPAS in combination with alkyltrichlorosilane-based self-assembled monolayers proved superior towards the detection of cocaine. The most efficient method for the attachment of the aptamers onto the sensor surface to construct highly dense populations of the aptamer molecules with retained biomolecule activity is shown to be dependent on the composition of immobilizing solution and on the amount of spacing provided in the plane of the aptamer molecules. The distinct ligand-induced binding mechanisms and regeneration capabilities of the two anti-cocaine aptamers are monitored with the EMPAS. Utilizing this sensor to monitor cocaine-aptamer interactions will serve as the first piezoelectric aptasensor for the detection of a small molecule.

This study embarks on exploiting the Thickness Shear Mode (TSM) acoustic wave sensor and the ElectroMagnetic Piezoelectric Acoustic Sensor (EMPAS) towards the study of aptamer-to-cocaine binding in a label-free direct approach. The high sensitivity and selectivity offered by the EMPAS in combination with alkyltrichlorosilane-based self-assembled monolayers proved superior towards the detection of cocaine. The most efficient method for the attachment of the aptamers onto the sensor surface to construct highly dense populations of the aptamer molecules with retained biomolecule activity is shown to be dependent on the composition of immobilizing solution and on the amount of spacing provided in the plane of the aptamer molecules. The distinct ligand-induced binding mechanisms and regeneration capabilities of the two anti-cocaine aptamers are monitored with the EMPAS. Utilizing this sensor to monitor cocaine-aptamer interactions will serve as the first piezoelectric aptasensor for the detection of a small molecule.

Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models.

Development of chiral ligands capable of achieving high selectivity for various asymmetric catalytic reactions has been an important aim of both academia and industry. Nature is capable to selectively catalyze chemical reactions by using enzymes. An ideal catalyst would combine the selectivity of nature and the reactivity of man-made catalysts based on transition metal complexes. The two biomolecules chosen to achieve this are DNA and PNA. DNA is a chiral molecule with high binding selectivity towards small molecules and has been used as ligand for asymmetric catalysis. PNA is an achiral structural analogue of DNA that can form duplexes with DNA. To produce DNA based catalysts it is necessary to introduce a ligand such as a phosphine that will strongly coordinate to transition metals. To achieve this, functionalized linkers need to be introduced into a DNA strand, to covalently couple the phosphine moiety at a specific location of the DNA strand. Amine linkers and several modified nucleosides have been prepared containing thiol and amine functionalities and some of them were successfully introduced into DNA strands to function as linkers for the introduction of phosphine functionalities. Those strands were purified and an adequate procedure was developed for their analysis by MALDI-TOF. Diphenylphosphino carboxylic acids have been coupled to amine modified deoxyuridines by amide bond formation. The same coupling method has been used for oligonucleotides. DNA strands containing phosphine moieties were characterized by MALDI-TOF and ³¹P NMR spectrometry. ³¹P NMR spectroscopy was also used to confirm coordination of a phosphine modified 15-mer to [PdCl(η³-allyl)]₂. The phosphine modified nucleobases were also tested as ligands for palladium catalyzed allylic alkylation and allylic amination with diphenylallyl acetate as substrate although no enantioselectivity was observed. A PNA monomer was also modified with a bidentate sulfur protected phosphine and successfully introduced into a short PNA strand using manual solid phase synthesis. This strand was analyzed by MALDI-TOF. Moreover, preliminary studies were performed to test the use of aptamers as scaffolds for targets containing a ligand functionality.

Hsp27 appartient à la famille des protéines dites de survie comme Bcl2 ou la survivine. C’est une protéine anti-apoptotique qui subit une dérégulation de son expression dans de nombreux types tumoraux. Elle est caractérisée comme étant une cible thérapeutique majeure. Au cours de ma thèse, j’ai isolé des peptides stabilisés, dit aptamères, capables d’inhiber fonctionnellement les activités anti-apoptotiques et tumorigènes d’Hsp27. Ces aptamères perturbent la biochimie structurale de Hsp27 et induisent le blocage du cycle cellulaire in vivo. Parallèlement à cette étude, j’ai caractérisé les effets de la déplétion de Hsp27 sur la formation de métastases et de tumeurs osseuses. J’ai aussi montré que la modification du taux de Hsp27 induisait la dégradation de différentes protéines, dites clientes, comme la caspase3, HDAC6 et STAT2. / Hsp27 belongs to the class of survival proteins like Bcl2 or survivin. This protein was well categorized as a major anti apoptotic protein as displaying a high level of expression in lot of tumor types. Moreover, Hsp27 is referenced as a major therapeutic target in cancer. During my PhD, I characterized stable peptides called aptamers, which functionally blocked Hsp27 antiapoptotic and tumorigenic properties. These aptamers disrupted biochemical and structural states of Hsp27 and promoted cell cycle arrest in xenografts. In the same time, I have characterized the effect of Hsp27 depletion in metastasis establishment and bone marrow tumor growth. I have shown that targeting level of Hsp27 induced degradation of several of its client proteins like caspase3, HDAC6 and STAT2

Des techniques variées de visualisation de molécules d’intérêt sur cellules vivantes ou fixées ont permis de suivre leur synthèse, localisation, dégradation et autres activités. Dans cette étude, nous avons développé deux outils de fluorescence pour étudier la synthèse des protéines sur bactéries vivantes. Le premier décrit l’utilisation du système Spinach pour l’imagerie du ribosome. Cette approche diffère des méthodes conventionnelles qui utilisent des protéines fluorescentes puisque l’ARN ribosomal 16S contient un aptamère qui rend fluorescent un composé fluorogène. Une étude comparative de la performance de différents aptamères Spinach a été réalisée. Un deuxième outil se focalise sur l’accumulation d’un antibiotique de la famille des aminoglycosides (ligand du ribosome) conjugué à un fluorophore. Ce nouveau conjugué, qui a conservé son activité bactéricide permet pour la première fois de visualiser l’accumulation de l’antibiotique sur bactérie vivante. Cela permet une analyse au niveau de la cellule unique d’une population bactérienne exposée à l’antibiotique. Nous avons également obtenu des données sur la localisation de l’antibiotique une fois qu’il a pénétré dans la bactérie à une résolution inégalée par microscopie super-résolutive. Nous espérons que ces deux méthodes vont maintenant permettre une meilleure compréhension de la synthèse des protéines et fournir une vue nouvelle de la pénétration des antibiotiques dans les bactéries pour y produire leur action bactéricide. / Various visualizing techniques have previously enabled monitoring the fate of molecules of interest: their expression, localization, degradation and other activities in live or fixed cells. In this study, we have developed two fluorescent tools to study protein synthesis in live bacterial cell. The first one describes the application of Spinach system to ribosomes imaging. This is different from conventional methods (that use fluorescent proteins) in that 16S rRNA contains an inserted RNA aptamer that elicits fluorescence of a fluorogenic compound. A comparative study of the performance of different Spinach aptamers was performed here. A second system focuses on the uptake of a fluorescently labeled ligand of the ribosome, an antibiotic of the class of aminoglycosides. This novel conjugate, which kept its bactericidal activity allows for the first time imaging of aminoglycoside uptake on live bacteria. This opened the door to a single cell analysis of bacterial cell populations. We also obtained data about the localization of the antibiotic once inside the bacteria to an unprecedented resolution using super resolution microscopy. We hope that both of these methods will contribute to a better understanding of protein synthesis as well as provide a novel view on the way antibiotics penetrate into cells and perform their bactericidal action.

CNT membrane platforms are biomimetic polymeric membranes imbedded with carbon nanotubes which show fast fluid flow, electric conductivity, and the ability to be grafted with chemistry. A novel micro-dialysis probe nicotine concentration sampling technique was proposed and proved in vitro, which could greatly improve the efficiency and accuracy of future animal transdermal studies. To enhance the scope of transdermal drug delivery which was limited to passive diffusion of small, potent lipophilic drugs, a wire mesh lateral electroporation design was also proposed which could periodically disrupt the skin barrier and enhance drug flux. It was shown that AMP binding aptamer at the tip of carbon nanotubes may act as gatekeepers and regulate ionic transport through CNT membrane. Multiple cycle gating of ionic transport upon AMP binding/unbinding which changes the aptamer conformation was displayed. This CNT membrane-aptamer system closely mimics how protein ion channels modulate ion flow by responding to stimuli, which may have significant impact on active membrane transport. Finally an enhanced electroosmosis concept by “ratchet” functionalization at both ends of carbon nanotubes in was discussed. Direct observation of water transport by electroosmosis was made possible through enhanced flow in vertically aligned high flux CNT membranes.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-09-13T16:28:44Z No. of bitstreams: 2 Dissertação - Evellyn Gonçalves de Souza - 2017.pdf: 2198173 bytes, checksum: 0e490bd8b1f7554afb334a3e27faf8ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-19T13:57:54Z (GMT) No. of bitstreams: 2 Dissertação - Evellyn Gonçalves de Souza - 2017.pdf: 2198173 bytes, checksum: 0e490bd8b1f7554afb334a3e27faf8ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-19T13:57:54Z (GMT). No. of bitstreams: 2 Dissertação - Evellyn Gonçalves de Souza - 2017.pdf: 2198173 bytes, checksum: 0e490bd8b1f7554afb334a3e27faf8ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Dengue is an endemic disease that causes wide concern both the health systems and to the patients. Thus, it is important that studies aiming at diagnoses faster and with possibility of field application can be developed for the medical intervention happens early, avoiding the worsening of the cases. Therefore, the aim of this work was to develop an electrochemical biosensor based on the use of aptamer as a bioelement for recognition on the surface of the boron doped diamond electrode (BDD) for the detection of each of the four serotypes of the dengue virus. Previously to the development of the biosensor, the dot-blot and apta-PCR techniques were used as validation methods for the interaction between aptamer B07 and target, the 5'UTR region present in the viral genome. The cyclic voltammetry technique was used in the analysis of redox reactions on the surface of the BDD electrode with and without modification of its surface with chitosan film. Ferrocene was used as the redox mediator and electroactive indicator of hybridization of the DNA strands formed in the sensor. The peaks of current indicated that the electrode with chitosan film modification on its surface presented greater stability. The redox compound had higher affinity for the double chains hybridized on the surface of the electrode, showing current values for DENV 1, 2, 3 and 4 of were 0.73; 0.69; 0.79 and 1.03 μA, respectively. These values were higher than the current found for the single-stranded aptamer (ssDNA), which was 0.62 μA, as well as for the current obtained from the aptamer hybridized with its complementary strand (dsDNA) whose value was 0.91 μA. Analyzes with time variations were performed showing a reduction in current values as a function of time, probably due to the reduction of the interaction of the electroactive material in the sensor. The aptasensor developed here showed good detection distinction between nucleic acid sequences, presenting potential for application in the detection of dengue virus. / A dengue é uma doença endêmica que causa grande preocupação tanto aos sistemas de saúde quanto aos pacientes. Dessa forma, é importante que estudos visando diagnósticos mais rápidos e com possibilidade de aplicação a campo possam ser desenvolvidos para que a intervenção médica possa ser precoce, evitando-se o agravamento dos casos. Neste sentido, buscou-se no presente trabalho desenvolver um biossensor eletroquímico baseado no uso de aptâmero como bioelemento reconhecedor na superfície do eletrodo de diamante dopado com boro (DDB) para a detecção de cada um dos quatro sorotipos do vírus da dengue. Previamente ao desenvolvimento do biossensor, as técnicas de dot-blot e apta-PCR foram utilizadas como métodos de validação da interação entre o aptâmero B07 e alvo, a região 5’UTR presente no genoma viral. A técnica de voltametria cíclica foi utilizada nas análises de reações redox na superfície do eletrodo DDB com e sem modificação de sua superfície com filme de quitosana. O composto ferroceno foi utilizado como mediador redox e indicador eletroativo de hibridização das cadeias de DNA formadas no sensor. Os picos de corrente indicaram que o eletrodo com modificação de filme de quitosana em sua superfície apresentou maior estabilidade, onde o composto redox teve maior afinidade pelas duplas cadeias hibridizadas na superfície do eletrodo, apresentando valores de correntes para DENV 1, 2, 3 e 4 de foram 0,73; 0,69; 0,79 e 1,03 μA, respectivamente. Esses valores foram superiores a corrente encontrada para o aptâmero em fita simples (ssDNA) que foi de 0,62 μA, assim como observada para corrente obtida do aptâmero hibridizado com sua fita complementar (dsDNA) cujo valor obtido foi de 0,91 μA. Análises com variações de tempo foram realizadas apresentando redução nos valores de correntes em função do tempo, provavelmente devido à redução da interação do material eletroativo no sensor. O aptasensor aqui desenvolvido apresentou boa detecção distinção entre sequências de ácidos nucleicos, apresentando potencial para aplicação na detecção do vírus da dengue.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-10-20T16:57:25Z No. of bitstreams: 2 Dissertação - Rívia Aparecida Reinalda Arruda - 2016.pdf: 3000238 bytes, checksum: 221f622157672c4f34ee32f2aed12d0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-10-21T19:20:24Z (GMT) No. of bitstreams: 2 Dissertação - Rívia Aparecida Reinalda Arruda - 2016.pdf: 3000238 bytes, checksum: 221f622157672c4f34ee32f2aed12d0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-21T19:20:24Z (GMT). No. of bitstreams: 2 Dissertação - Rívia Aparecida Reinalda Arruda - 2016.pdf: 3000238 bytes, checksum: 221f622157672c4f34ee32f2aed12d0b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main purpose of this study was to assess conformational changes in preselected aptamers A03 and C10 against untranslated regions present in the genome of the dengue virus (DENV), by means of molecular biology and physics assays. Aptamers are nucleic acid molecules with potential theranostic applications. In this context, dengue was the target chosen for study because is an important infectious viral disease presenting high rates of morbidity and mortality in the worldwide, especially in tropical and subtropical areas, where there are high incidences of the mosquito vector Aedes aegypti. These aptamers showed, by dot-blot and magnetic beads ligand capture and subsequent qPCR detection, similar affinities and specificities to binding to the DENV 5'UTR. Probably, the analogy of the motifs and secondary structures presented in these ligands, identified by in silico studies, are responsible for these similarities. Data obtained by using Fluorescence Resonance Energy Transfer Assays (FRET) confirmed the affinity of A03 as DENV ligand by conformational changes in the double labeled aptamer (5’-FAM, 3’-TAMRA). Aptamer conformacional changes resulting from its interaction with the target were detected by the modifications in the fluorescence intensities between FAM and TAMRA fluorophores. These conformational changes results in donor-acceptor distance modification that has an effect on FRET efficiency and on fluorescence intensity of these molecules. The ionic strength of the buffer used in the trials had significant influence on the conformation of the aptamer, allowing the detection of interactions between A03 and DENV. The ions in the buffer lead an effect in the conformation of the aptamer, increasing its thermal stability, and the Mg2+ ion has led to major conformational changes on A03 compared to Na+ ion. The data presented herein demonstrated the importance of the studies of oligonucleotides binding affinity and specificity to Dengue virus. It can be concluded that the A03 and C10 aptamers have potential in application for diagnostic and/or therapeutic purposes, considering the diluent buffer, since different salts have influence on the secondary and tertiary conformational structures of aptamers, and on bioavailability of interaction with the target. / O objetivo principal desse trabalho foi avaliar alterações conformacionais nos aptâmeros A03 e C10 previamente selecionados contra regiões não traduzidas presentes no genoma do vírus da dengue (DENV), por meio de testes de biologia e física molecular. Os aptâmeros são moléculas de ácidos nucleicos com potencial aplicação na teranóstica das mais diversas doenças. Nesse contexto, destaca-se a dengue, uma doença infecciosa viral com altos índices de morbi-mortalidade em um cenário mundial, principalmente em regiões tropicais e subtropicais, onde são altas as incidências do mosquito vetor Aedes aegypti. Esses aptâmeros demonstraram, por meio de dot blot e de captura de ligantes por beads magnéticos com detecção por PCR em Tempo Real, afinidades e especificidades semelhantes de ligação à 5’UTR do DENV. Provavelmente, essas semelhanças se deram em função dos motivos e das estruturas secundárias análogas apresentadas por esses ligantes, obtidas por ensaios in silico. Os ensaios de Transferência Ressonante de Energia de Fluorescência (FRET) permitiram a confirmação dessa afinidade de ligação entre A03:DENV, devido às alterações estruturais ocorridas no aptâmero duplamente marcado em sua sequência (5’-FAM; 3’-TAMRA). As alterações conformacionais no aptâmero resultantes de sua interação com o alvo foram detectadas pelas mudanças nas intensidades de fluorescência do FAM e do TAMRA, decorrentes de alterações nas distâncias entre o doador e o aceitador que afetam a eficiência FRET e por conseguinte as intensidades de fluorescência destas moléculas. A força iônica do tampão utilizado nos ensaios apresentou importante influência na conformação do aptâmero, permitindo a detecção das interações entre A03 e o DENV. Os íons presentes na solução tampão afetaram a conformação estrutural do aptâmero e aumentaram sua estabilidade térmica, sendo que o íon Mg2+ levou a maiores alterações estruturais sobre o A03 comparado ao íon Na+. Os dados aqui apresentados demostraram a importância dos estudos de afinidade e especificidade dos oligonucleotídeos ligantes ao vírus da dengue. É possível concluir que os aptâmeros A03 e C10 apresentam potencial de aplicação para fins diagnósticos e/ou terapêuticos, considerando o tampão diluente, uma vez que os diferentes sais presentes exercem influência sobre as estruturas conformacionais secundárias e terciárias dos aptâmeros, e em sua biodisponibilidade de interação com o alvo.

An ideal biosensor is a compact and in-expensive device that is able to readily and rapidly detects different types of analytes with high sensitivity and specificity. The affectability of a biosensing methodology is subject to the limit of nanomaterials to transduce the target binding process to an improved perceptible signal, while the selectivity is accomplished by considering the binding and specificity of certain moieties to their targets. Keeping these requirements in mind we have chosen nanomaterials such as carbon nanotubes (CNTs) and gold nanoparticles (AuNPs) that has catalytic properties combined with their size, shape and configuration dependent chemical and physical properties as essential precursors and signaling components for creation of biosensors with tremendous sensitivity. The primary goal of the research work described in this dissertation is to develop and evaluate novel methods to detect various analytes using nanomaterials, at the same time making an affordable architecture for point-of-care (POC) applications. We report here in chapter 3 a simple and new strategy for preparing disposable, paper-based, porous AuNP/M-SWCNT hybrid thin gold films with high conductivity, rapid electron transfer rates, and excellent electrocatalytic properties to achieve multiple analyte electrochemical detection with a resolution that greatly exceeds that of purchased flat gold slides. We further explored the use of nanomaterial-based paper films in more complex matrices to detect analytes such as NADH, which can act as a biomarker for certain cellular redox imbalances and disease conditions. Carbon nanotubes with their large activated surfaces and edge-plane sites (defects) that are ideal for performing NADH oxidation at low potentials without any help of redox mediators minimizing surface fouling in complex matrices is described in chapter 4. With an instrument-free approach in mind we further focused on a colorimetric platform using split cocaine aptamers and gold nanoparticles (AuNPs) to detect cocaine for on-site applications as described in chapter 5. In chapter 5, the split aptamer sequences were evaluated mainly on three basic criteria, the hybridization efficiency, specificity towards the analyte (cocaine), and the reaction time to observe a distinguishable color change from red to blue. The assay is an enzyme-assisted target recycling (EATR) strategy following the principle that nuclease enzyme recognizes probe–target complexes, cleaving only the probe strand releasing the target for recycling. We have also studied the effect of the number of binding domains with variable chain lengths on either side of the apurinic (AP) site. On the basis of our results, we finally shortlisted the sequence combination with maximum signal enhancement fold which is instrumental in development of colorimetric platform with faster, and specific reaction to observe a distinctive color change in the presence of cocaine.