Spelling suggestions: "subject:"7molecular probes."" "subject:"7molecular grobes.""
51 |
Conception et synthèse de sondes moléculaires pour l'étude d'interactions polyphénol-protéine / Design and synthesis of molecular probes for studying polyphenol-protein interactionsTran, Dong tien 18 December 2015 (has links)
Les polyphénols sont des métabolites secondaires d’origine végétale. Ces substances naturelles connues pour leurs pouvoirs antioxydants et anti-radicalaires, contribuent à la protection de la santé humaine notamment contre les maladies cardiovasculaires et neurodégénératives, mais également contre certains cancers et diabètes. Dans certains cas, ces effets biologiques bénéfiques pour la santé pourraient également être liés à une interaction spécifique polyphénol-protéine peu étudiée à ce jour par manque d’outils moléculaires adaptés. Les travaux effectués au cours de cette thèse ont consisté à concevoir, à synthétiser et à évaluer des sondes moléculaires polyvalentes porteuses de polyphénols comme substrats d’affinité pour l’analyse des interactions polyphénol-protéine. Dans ce contexte, de nombreuses sondes arborant différents types de polyphénols ont été synthétisées. Ces différentes sondes pourront être utilisées en protéomique chimique du type "Affinity-Based Protein Profiling" (ABPP) pour identifier au sein d’un mélange complexe de protéines, une protéine ayant une affinité spécifique pour un polyphénol donné. Ces mêmes sondes permettront également d’étudier de manière qualitative les interactions d’un polyphénol avec une protéine donnée en temps réel par la technique de résonance plasmonique de surface (SPR). / Polyphenols are plant secondary metabolites. These natural substances, known for their antioxidant and anti-free radical properties, generally contribute to the protection of human health not only against cardiovascular and neurodegenerative diseases, but also against certain cancers and diabetes. In some cases, these beneficial biological effects could also be related to specific polyphenol-protein interactions. However, studying this type of interactions has suffered from the lack of adequate molecular tools. The work carried out during this thesis has included designing, synthesizing and evaluating modulable polyfunctional molecular probes carrying polyphenols as affinity substrates to analyze polyphenol-protein interactions. In this context, various probes harboring different kinds of polyphenols were synthesized. These probes could be used in chemical proteomics following an “Affinity-Based Protein Profiling” approach (ABPP) to identify a protein within complex protein mixtures, which has a specific affinity with a given polyphenol. These probes will also allow studying the interactions of a polyphenol with a given protein in real time in a qualitative way by surface plasmon resonance (SPR).
|
52 |
Synthèse de sondes moléculaires pour l'imagerie multimodale et multi-échelle appliquée en science du vivant / Molecular probes synthesis for multimodal and multi-scale imaging in life scienceVorng, Jean-luc 16 December 2013 (has links)
Les travaux décrits dans ce mémoire ont pour objectif d'imager des cellules par des techniques d'imagerie complémentaires à la spectroscopie de fluorescence : la micro-spectroscopie Raman et l'imagerie NanoSIMS. Le présent manuscrit se divise en quatre chapitres et présente les différents aspects menant à la mise en place de ce projet. Le premier chapitre du manuscrit a pour but de positionner le projet d'un point de vue biologique. Ce chapitre présente l'organite que nous souhaitions marquer, le nucléole, et la façon dont nous avons procédé à sa localisation par l'intermédiaire de sondes moléculaires. Le marquage que nous avons choisi est un immuno-marquage indirect faisant intervenir deux anticorps : un anticorps primaire spécifique à la nucléophosmine, protéine majoritairement présente dans le nucléole et un anticorps secondaire, reconnaissant l'anticorps primaire, marqué par une sonde moléculaire d'intérêt. Ce chapitre a également permis de montrer par une analyse en immunofluorescence l'absence d'interférence des sondes moléculaires dans le cadre de ce marquage. Le second chapitre se focalise sur la synthèse des sondes moléculaires permettant le marquage des anticorps secondaires. Les sondes envisagées pour ce marquage possèdent un groupement ester de succinimide réactif capable de "s'accrocher" de manière covalente aux fonctions amines de l'anticorps secondaire et un ou plusieurs groupements fonctionnels visible par la technique d'imagerie voulue. Trois catégories de sondes ont été préparées et dépendent de la technique d'imagerie employée. Les sondes Raman comportant une triple liaison carbone-carbone visible par cette technique, les sondes NanoSIMS dont l'acquisition d'image sera possible par les halogènes présents dans la structure et les sondes bimodales comportant les deux éléments dans la même structure. Les sondes bimodales sont obtenues par le couplage pallado-catalysé de Sonogashira. Dans la dernière partie du chapitre, une nouvelle série de sonde a été envisagée dans le cadre d'une application future : le marquage de l'ATP-ϒ-SH grâce à une fonction mésylate. Les deux derniers chapitres ont pour but de mettre en application tout ce qui a été présenté dans les chapitres précédents. Les deux techniques employées permettent d'accéder à des types d'informations différentes : la spectroscopie Raman donne accès aux modes de vibrations d'une molécule tandis que l'imagerie NanoSIMS permet d'obtenir des informations élémentaires et isotopiques. Nous présenterons le cheminement suivi pour imager des cellules via leurs constituants par micro-spectroscopie Raman et imagerie NanoSIMS via les sondes moléculaires introduites. / Life sciences imaging are widely used for different applications, they are interested in medical diagnosis as well as basic research. In cells biology, fluorescence microscopy is mainly used for organelles observation at sub-cellular scale. However, techniques based on fluorescence phenomena are limited by some drawbacks like technical resolution, fluorescent dye degradation and the number of channels, which can be visualized. In this context, the exploration and the development of new way for image acquisition are considered as an experimental and technical scientific challenge. Furthermore, it can lead to complementary technique to fluorescence microscopy.This PhD thesis is a life science imaging project development and application allowing image acquisition base on molecular vibrations phenomena and elementary analysis in cells. Two techniques have been chosen in relation to both specificity: micro Raman spectroscopy and NanoSIMS imaging. Micro Raman spectroscopy allows the observation of molecular vibration mode at micron scale and NanoSIMS leads to elementary and isotopic sample information at sub-micronic scale. Combination of both techniques will lead to multi-scale and multi-modal imaging of biological samples. Molecular probes designing and synthesis for both techniques were used to visualize an organelle inside the nucleus: the nucleolus. Nucleolus has a key role in ribosomal RNA transcription and researchers shows some interest in the study of this organelle for his multifunctional role like ribosome biogenesis and nuclear organization. An immuno-labelling method combine with the introduction of molecular probes will allow nucleolus imaging by micro-Raman spectroscopy and NanoSIMS spectrometry. This immuno-labelling is specific to a phosphoprotein mainly localized inside the nucleolus: the nucleophosmin (NPM). In this project, the introduction of molecular probes in an immuno-labelling will act as a Raman Tag or a NanoSIMS tag for NPM's nucleolus observation and studies.This work at the interface between different fields: chemistry, biology and physics shows all the aspect of this project starting from molecular probes synthesis, immuno-labelling methods uses to direct application of both Raman and NanoSIMS techniques.
|
53 |
Localização e função de quinase de adesão focal e Calsarcina-1 em cardiomiócitos de ratos = emprego de sondas moleculares e lentivírus / Localization and function of focal adhesion kinase and Calsarcin-1 in rat cardiomyocytes : using of molecular probes and lentivirusConsonni, Sílvio Roberto, 1986- 05 August 2015 (has links)
Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T14:15:11Z (GMT). No. of bitstreams: 1
Consonni_SilvioRoberto_D.pdf: 7954683 bytes, checksum: 766ce32e5bbb5a5c0b8cc78fd2735dc0 (MD5)
Previous issue date: 2015 / Resumo: Há um crescente avanço no desenvolvimento das tecnologias que permitam a localização de proteínas em células por microscopias de luz e eletrônica combinadas, com o uso das sondas moleculares miniSOG e Apex2, por exemplo. Adicionalmente busca-se compreender como proteínas são responsáveis pelas funções celulares e teciduais. A Quinase de Adesão Focal (FAK), proteína da cascata de sinalização das integrinas é considerada uma mediadora em potencial do estresse mecânico nos cardiomiócitos. Sabe-se que em cardiomiócitos submetidos a estímulos hipertróficos, ocorre rápida ativação da FAK e sua redistribuição subcelular, contudo são pouco conhecidos os mecanismos envolvidos nesses processos. Outra proteína de grande importância no coração é a Calsarcina-1 (CS1), regulador negativo da via de Calcineurina, crucial no desenvolvimento da hipertrofia cardíaca. Entretanto os mecanismos envolvidos nessa regulação negativa, assim como a distribuição subcelular de CS1 são pouco conhecidos. Visando à interação entre essas áreas para estudo da distribuição espaço-temporal dos componentes celulares e de proteínas, bem como a importância da FAK e CS1 no sarcômero e seu papel na sinalização hipertrófica sob estímulo mecânico, o objetivo geral desse trabalho foi explorar a capacidade das proteínas FAK e CS1 para incorporar geneticamente sondas moleculares que permitissem monitorar por meio de imagens o comportamento dessas moléculas-chave na biologia do disco Z em MVRNs submetida ao estiramento mecânico. Para isso, foram realizados ensaios de localização subcelular com uso de sondas moleculares aplicadas à microscopia correlativa, bem como ensaios bioquímicos, moleculares e de atividade enzimática. Os dados confirmaram a FAK associada às fibras de actina e adesões focais em células H9c2 e foi demonstrado à microscopia de luz que FAK wild-type (wt) translocou-se parcialmente para o compartimento nuclear após estimulação do agonista fenilefrina, enquanto que FAK Y397F (forma mutante inativa) não apresentou mesmo fenótipo. Por outro lado, apesar da padronização e expressão das construções com FAK e miniSOG ou Apex2 em células HEK 293T e H9c2, não foi conclusiva a localização subcelular da FAK, por meio do uso de microscopia eletrônica em MVRN. Provavelmente devido à distribuição difusa da maior parte das moléculas de FAK, não foi identificada uma região elétron-densa conclusiva à microscopia eletrônica de transmissão. No tocante à importância da CS1, observou-se que o estiramento cíclico não induziu o aumento na expressão proteica ou gênica relativa de CS1 e CnA, assim como não houve alteração na atividade fosfatase de CnA. No entanto houve redução da interação de CS1 e CnA, bem como alteração na localização de CS1 em MVRN sob estímulo mecânico. Dados de superexpressão e silenciamento de CS1 corroboram a regulação negativa de CS1 à CnA em MVRN sob estímulo mecânico. Baseando-se em dados estruturais, especulou-se que como o sítio de ligação de NFAT e CS1 à CnA são muito próximos e ao mesmo tempo distante do sítio ativo da fosfatase, é possível que o papel de CS1 na regulação negativa de CnA ocorra por impedimento espacial ao fator de transcrição NFAT. Portanto, esses resultados podem contribuir para uma possível inferência farmacológica, visto que a via de Calcineurina-NFAT é uma das principais mediadoras de hipertrofia em cardiomiócitos, mediante estímulos patológicos / Abstract: There is an increasing move towards the development of technologies that allow the localization of proteins in cells by combined electron and light microscopy, with the use of molecular probes such as miniSOG and APEX2. Additionally we seek to understand how proteins are responsible for the cellular and tissue functions. The Focal Adhesion Kinase (FAK) is protein of integrin signaling cascade considered as a potential mediator of mechanical stress in cardiomyocytes. It is known that in cardiomyocytes subjected to hypertrophic stimuli by rapid activation of FAK and its subcellular redistribution, however the mechanisms involved in these processes are poorly understood. Another very important protein in the heart is Calsarcin-1 (CS1), a negative regulator of the Calcineurin pathway which is crucial in the development of cardiac hypertrophy. However the mechanisms involved in the negative regulation as well as the subcellular distribution CS1 are poorly understood. Aiming at the interaction between these areas to study the spatial-temporal distribution of cellular and protein components, and the importance of FAK and CS1 in the sarcomere and its role in hypertrophic signaling under mechanical stimulation, the aim of this study was to explore the ability of FAK and CS1 to incorporate genetically molecular probes that allow monitoring through images the behavior of these key molecules in the Z disc biology in MVRNs subjected to mechanical stretch. To this end, we performed subcellular localization assays using molecular probes applied to the correlative microscopy, biochemical and molecular assays and enzymatic activity. These data confirm the FAK associated with actin and focal adhesions fibers in H9c2 cells and has been shown by light microscopy that FAK wild-type (wt) is partially translocated to the nuclear compartment after stimulation of the agonist phenylephrine, while FAK Y397F (inactive mutant form) did not show the same phenotype. Moreover, despite standardization and expression of FAK and miniSOG or APEX2 in HEK 293T cells and H9c2, it was inconclusive subcellular localization of FAK, through the use of electron microscopy, in MVRN. Probably due to the diffuse distribution of most FAK molecules, it has no conclusive electron-dense region in transmission electron microscopy. Regarding the importance of CS1, it was observed that the cyclic stretch did not induce an increase in protein expression or gene relative CS1 and CnA, as there was no change in the phosphatase activity of CnA. However there was less interaction CS1 and CnA and change in CS1 location in MVRN under mechanical stimulation. CS1 overexpression and silencing corroborate the negative regulation of the CS1 over CnA in MVRN under mechanical stimulation. Based on structural data, it has been speculated that as the NFAT and CS1 binding sites are very close in CnA and at the same time distant from the active site of the phosphatase, it is possible that the role of CS1 in the negative regulation of CnA occur by steric hindrance to the NFAT transcription factor. Therefore, these results may contribute to a possible pharmacological inference, whereas Calcineurin-NFAT pathway is a major mediator of hypertrophy in cardiac myocytes by pathologic stimuli / Doutorado / Biologia Tecidual / Doutor em Biologia Celular e Estrutural
|
54 |
Synthesis and Characterization of Novel Flavonoid-Based Fluorescent Sensors and other Sensors with Excited State Intramolecular Proton Transfer for Biological ApplicationsMcDonald, Lucas J. 23 May 2018 (has links)
No description available.
|
55 |
Nanoparticle Probes for Ultrasensitive Biological Detection and Motor Protein Tracking inside Living CellsAgrawal, Amit 09 November 2006 (has links)
Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)
|
56 |
Novel probes, carriers and prodrugs to target senescent cells in vivoLozano Torres, Beatriz 02 September 2021 (has links)
[ES] La presente tesis doctoral titulada "Nuevas sondas, portadores y pro-fármacos dirigidos a células senescentes in vivo" se centra en el diseño, preparación, caracterización y evaluación de sondas basadas en fluoróforos funcionalizados o en nanopartículas, así como en el desarrollo de profármacos, aplicadas al campo de la senescencia celular.
En el primer capítulo se introducen, a nivel general, los diferentes conceptos relacionados con el reconocimiento molecular y la detección de biomarcadores. Seguidamente se introducen conceptos básicos de senescencia celular y envejecimiento, así como el papel que juegan las células senescentes a nivel fisiológico, en enfermedades asociadas al envejecimiento celular y en el cáncer. Por último, se aborda también la creciente necesidad de desarrollar nuevas herramientas de diagnóstico y terapias centradas en la detección y tratamiento de dichas enfermedades.
En el segundo capítulo se exponen los objetivos generales de la presente tesis doctoral, así como los objetivos concretos que son abordados en los diferentes capítulos experimentales.
En el tercer capítulo se describe una nueva sonda molecular para la detección de senescencia in vivo. En concreto, se describe el diseño de una sonda de dos fotones basada en un fluoróforo de naftalimida conectada, mediante un éster metílico de L-histidina, con una galactosa acetilada que esta unida a uno de los átomos de nitrógeno aromático de la L-histidina a través de un enlace N-glicosídico hidrolizable (AHGa). La sonda inicial presenta una baja emsión pero en células senescentes se transforma en un fluoróforo con alto rendimiento cuantico que permite visualizar estas células debido a la hidrólisis del enlace N-glicosidico por la enzima ¿-galactosidasa sobreexpresada en este tipo de células. La sonda es capaz de detectar in vitro células de melanoma humano SK-Mel-103 tratadas con
quimioterapia inductora de senescencia frente a células control. Adicionalmente, se validó la detección in vivo de senescencia en ratones con xenoinjertos tumorales tratados con quimioterapia inductora de senescencia.
Basándonos en los resultados obtenidos en el capítulo tres, en el capítulo cuatro se describe un sistema similar cuyas características confieren al fluoróforo una mayor longitud de onda de excitación (HeckGal). La nueva sonda es capaz de detectar células senescentes debido a la sobreexpresión de la enzima ¿-galactosidasa asociada a senescencia en gran variedad de líneas celulares con diversos métodos de inducción de senescencia. Esta sonda se validó también in vivo en un modelo ortotópico de cáncer de mama de ratón tratado con quimioterapia inductora de senescencia, y en un modelo de ratón de fibrosis renal.
Seguidamente, el capítulo cinco, se centra en un nuevo concepto de sondas moleculares no invasivas, que proporcionan una señal fácilmente legible a través de simples medidas de fluorescencia de la orina. Esta idea se llevó a cabo mediante la funcionalización de una sonda con grupos que le confieren características diuréticas (rápida eliminación renal) y el concepto se aplicó al diseño de una sonda para a la detección en orina de la carga senescente en diversos modelos. La sonda (Cy7Gal) está basada en la cianina-7 que es hidrolizada por la enzima ¿-galactosidasa en un fluoróforo altamente emisivo Cy7. Cy7Gal y Cy7 contienen restos de ácido sulfónico que aumentan su solubilidad en agua y desencadenan su rápida excreción por el sistema urinario. El grado de senescencia se cuantificó por medición directa de fluorescencia en orina en tres modelos de senescencia en ratones: (i) un modelo ortotópico de cáncer de mama en ratones BALB/cByJ con senescencia inducida por quimioterapia; (ii) ratones BALB / cByJ envejecidos de forma natural; y (iii) ratones con senescencia acelerada (SAMP8, del inglés Senescence Accelerated Mouse-Prone 8). Además, las imágenes IVIS ex vivo permite / [CA] La tesi doctoral titulada "Noves sondes, portadors i profàrmacs dirigits a cèl·lules senescents in vivo" se centra en el desenvolupament i avaluació de sondes basades en fluoròfors funcionalitzats o en nanopartícules, així com en el desenvolupament de profàrmacs, aplicades a el camp de la senescència cel·lular.
Primer s'introdueixen els diferents conceptes relacionats amb el sensing i amb la sescencia cel·lular, abordant la necessitat de noves eines diagnòstiques i terapèutiques d'aquestes malalties. En els capítols 3:04 es descriuen una nova sonda molecular dos fotons per a la detecció de senescència in vivo en ratolins amb xenoempelts tumorals tractats amb quimioteràpia inductora de senescència, una altra sonda per a la detecció in vivo en un model ortotòpic de càncer de mama de ratolí tractat amb quimioteràpia inductora de senescència, i en un model de ratolí de fibrosi renal. El capítol 5, se centra en un nou concepte de sondes moleculars no invasives, que proporcionen un senyal fàcilment llegible a través de simples mesures de fluorescència de l'orina. Finalment, pel que fa a detecció de senescència cel·lular es refereix, es presenta en el capítol sis la detecció in vivo de senescència cel·lular mitjançant l'alliberament controlat de Nile Blue en nanopartícules de sílice mesoporoses funcionalitzades amb un galactohexasacárido. Un cop assolits els objectius planificats pel que fa a la diagnosi, en el capítol set es va abordar la millora d'un dels senolíticos més potents i àmpliament conegut en el camp de la senescència cel·lular que existeix a dia d'avui al mercat: el Navitoclax. / [EN] The doctoral thesis entitled "New probes, carriers and pro-drugs directed to senescent cells in vivo" focuses on the development and evaluation of probes based on functionalized fluorophores or nanoparticles, as well as on the development of prodrugs, applied to the field of cellular senescence.
First, the different concepts related to sensing and cell sescence are introduced, addressing the need for new diagnostic and therapeutic tools for these diseases. Chapters three and four describe a new two-photon molecular probe for in vivo senescence detection in mice with tumor xenografts treated with senescence-inducing chemotherapy, another probe for in vivo detection in an orthotopic mouse breast cancer model. treated with senescence-inducing chemotherapy, and in a mouse model of renal fibrosis. Chapter five focuses on a new concept of non-invasive molecular probes, which provide an easily readable signal through simple measurements of urine fluorescence. Finally, regarding the detection of cellular senescence, chapter six presents the in vivo detection of cellular senescence through the controlled release of Nile Blue in mesoporous silica nanoparticles functionalized with a galactohexasaccharide. Once the planned objectives with regard to diagnosis had been achieved, chapter seven addressed the improvement of one of the most powerful and widely known senolytics in the field of cellular senescence that exists on the market today: Navitoclax. / The authors thank the financial support from the Spanish Government (projects MAT2015-64139-C4-1-R and AGL2015-70235-C2-2-R) and the
Generalitat Valenciana (project PROMETEOII/2014/047). R.M laboratory members thank the financial support from the Spanish Government (project RTI2018-100910-B-C41) and the Generalitat Valenciana (project PROMETEO 2018/024). B.L-T. is grateful to the Spanish Ministry of Economy for their PhD grants (FPU15/02707). Work in the laboratory of MS was funded by the IRB and by grants from the Spanish Ministry of Economy co-funded by the European Regional Development Fund (ERDF) (SAF2013-48256-R), the European Research Council (ERC-2014-AdG/669622), and the “laCaixa” Foundation. D.M.-E. laboratory is supported by the Cancer Research UK (CRUK) Cambridge Centre Early Detection Programme, by a CRUK Early Detection OHSU Project Award (C62187/A26989), by a Medical Research Council (MRC) New Investigators Research Grant (NIRG, MR/R000530/1). Work in the laboratory of I.F. is supported by grants from the Spanish Government (SAF2017-86690-R), Instituto de Salud Carlos III (CIBERNED and RETIC Tercel), and Generalitat Valenciana (Prometeo 2017/030). / Lozano Torres, B. (2021). Novel probes, carriers and prodrugs to target senescent cells in vivo [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172175
|
57 |
Development of Smart Devices for the Detection of Metabolites of Toxic Substances and Disease-Related Enzyme OverexpressionDomínguez Rodríguez, Marcia 18 January 2024 (has links)
Tesis por compendio / [ES] Esta tesis se enfoca en el desarrollo de dispositivos inteligentes para detectar metabolitos de sustancias tóxicas y la sobreexpresión enzimática relacionada con enfermedades. Se aborda la limitación de las técnicas de diagnóstico convencionales y se destaca la utilidad de la orina como muestra biológica. Se discuten conceptos de materiales mesoporosos de sílice, reconocimiento molecular y sondas moleculares fluorogénicas. Los objetivos se detallan, y luego se presenta un nanodispositivo para detectar ácido trans,trans-mucónico (t,t-MA) en orina (S4). Se describe una sonda fluorogénica en el infrarrojo cercano para la detección de elevados niveles de alanina aminopeptidasa en orina (NB-ALA). También se presentan nuevas sondas para detectar biomarcadores de cáncer (NB-SO3-Leu y NB-SO3-Ala), resaltando la importancia de su solubilidad y eliminación renal. Finalmente, se propone una sonda molecular no invasiva (Cy7-MAO) para detectar la enzima monoamina oxidasa a través de medidas de fluorescencia en la orina. Las conclusiones subrayan el potencial de estos sistemas para el diagnóstico y tratamiento de enfermedades. / [CA] Aquesta tesi s'enfoca en el desenvolupament de dispositius intel·ligents per detectar metabòlits de substàncies tòxiques i la sobreexpressió enzimàtica relacionada amb malalties. S'aborda la limitació de les tècniques de diagnòstic convencionals i destaca la utilitat de l'orina com a mostra biològica. Es discuteixen conceptes de materials mesoporosos de sílice, reconeixement molecular i sondes moleculars fluorogèniques. Els objectius es detallen, i després es presenta un nanodispositiu per detectar àcid trans, transmucònic (t, t-MA) en orina (S4). Es descriu una sonda fluorogènica a l'infraroig proper per a la detecció d'elevats nivells d'alanina aminopeptidasa en orina (NB-ALA). També es presenten noves sondes per detectar biomarcadors de càncer (NB-SO3-Leu i NB-SO3-Ala), ressaltant la importància de la seva solubilitat i eliminació renal. Finalment, es proposa una sonda molecular no invasiva (Cy7-MAO) per detectar l'enzim monoamina oxidasa a través de mesures de fluorescència a l'orina. Les conclusions subratllen el potencial d'aquests sistemes per al diagnòstic i el tractament de malalties. / [EN] This PhD thesis focuses on the development of smart devices for detecting metabolites of toxic substances and enzyme overexpression related to diseases. It addresses the limitations of conventional diagnostic techniques and highlights the usefulness of urine as a biological sample. Concepts of mesoporous silica materials, molecular recognition, and fluorogenic molecular probes are discussed. The objectives are outlined, and then a nanodevice for detecting trans, trans-muconic acid (t,t-MA) in urine (S4) is presented. A near-infrared fluorogenic probe for the detection of high levels of alanine aminopeptidase in urine (NB-ALA) is described. New probes for detecting cancer biomarkers (NB-SO3-Leu and NB-SO3-Ala) are also introduced, emphasizing the importance of their solubility and renal elimination. Finally, a non-invasive molecular probe (Cy7-MAO) is proposed for detecting monoamine oxidase enzyme through fluorescence measurements in urine. The conclusions underscore the potential of these systems for the diagnosis and treatment of diseases. / Domínguez Rodríguez, M. (2023). Development of Smart Devices for the Detection of Metabolites of Toxic Substances and Disease-Related Enzyme Overexpression [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/202015 / Compendio
|
58 |
Validation de modalités d’imagerie innovantes de l’athérosclérose par cathéter intravasculaire bimodal combinant fluorescence (NIRF) et ultrasons (IVUS) couplé à l’injection locale de sondes moléculaires in vivo ciblant ICAM-1 et le collagène.Bertrand, Marie-Jeanne 12 1900 (has links)
No description available.
|
59 |
CaracterizaÃÃo molecular e biolÃgica de um begomovÃrus isolado de tomateiro, Lycopersicon esculentum Mill., no estado de GoiÃs e sua interaÃÃo com o vetor Bemisia argentifolii Bellows & Perring / Molecular and biological characterization of a begomovirus isolated from tomato, Lycopersicon esculentum Mill. in the state of GoiÃs and its interaction with the vector Bemisia argentifolii Bellows & PerringCarmem Dolores Gonzaga Santos 00 July 2001 (has links)
CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os begomovÃrus, vÃrus da famÃlia Geminiviridae transmitidos por mosca branca, tÃm emergido como sÃrios patÃgenos de culturas agronÃmicas e hortÃcolas em regiÃes tropicais e subtropicais de muitos paÃses em todo o mundo. A partir da dÃcada de 80, tÃm aumentado os relatos da disseminaÃÃo da mosca branca, Bemisia argentifolii, e de begomovÃrus provocando impacto devastador nas regiÃes em que ocorrem. No Brasil, estes patÃgenos tÃm sido limitantes para a produÃÃo de tomate (Lycopersicon esculentum) em vÃrias Ãreas de cultivo com incidÃncia crescente desde 1994. No presente trabalho, plantas de tomate exibindo sintomas de infecÃÃo provocada por vÃrus como mosaico, clorose internerval, enrolamento do limbo foliar e reduÃÃo do crescimento, foram coletadas em lavouras de tomate indÃstria em AnÃpolis-GO. O vÃrus foi identificado como pertencente ao gÃnero Begomovirus mediante tÃcnica de PCR usando oligonucleotÃdeos especÃficos que amplificaram fragmentos dos componentes A e B do genoma viral. No capÃtulo I sÃo apresentados os resultados da caracterizaÃÃo molecular e no capÃtulo II, os dados da determinaÃÃo do cÃrculo de hospedeiros e da investigaÃÃo da relaÃÃo do begomovÃrus com o vetor Bemisia argentifolii. O isolado denominado GOANPL, foi clonado e parcialmente seqÃenciado tendo sido obtidas as seqÃÃncias nucleotÃdicas dos genes da capa proteica, Rep e de toda a regiÃo intergÃnica, em um total de 2.130 nucleotÃdeos. A anÃlise comparativa das seqÃÃncias revelou que, em geral, o GOANPL possui relacionamento genÃtico distante com begomovÃrus da Ãsia, Europa e Ãfrica sendo mais prÃximo das espÃcies do Brasil, particularmente, com os begomovÃrus identificados em Minas Gerais (TRMV) e no Distrito Federal (DF-BR2). Com este Ãltimo, apresentou alta homologia em todo o genoma podendo vir a constituir, com o mesmo, uma nova espÃcie. A determinaÃÃo do cÃrculo de hospedeiros do GO-ANPL foi realizada inoculando-se 46 espÃcies vegetais pertencentes a nove famÃlias botÃnicas, sob duas modalidades de inoculaÃÃo: mecÃnica e com a mosca branca. Constatou-se que o GO-ANPL infecta, preferencialmente, plantas da famÃlia Solanaceae como Nicotiana benthamiana, Datura stramonium e Nicandra physalodes. O nÃmero de espÃcies infectadas com o inseto vetor foi superior ao obtido pela inoculaÃÃo mecÃnica e diferiu dos resultados obtidos para outros isolados de begomovÃrus de tomate no Brasil. Os testes foram todos confirmados com hibridizaÃÃo com sondas moleculares, em \"dot blot\"No estudo da relaÃÃo vÃrus-vetor, foram investigados o perÃodo de acesso de aquisiÃÃo do vÃrus (PAA), o perÃodo de acesso de inoculaÃÃo do vÃrus (PAI), e o perÃodo de latÃncia do vÃrus na fase adulta do vetor, empregando-se cinco moscas/planta de tomate \'Santa Clara\' em todos os tratamentos. Para a definiÃÃo do PAA e do PAI, foram testados nove diferentes perÃodos de tempo: 0,25, 0,5, 1, 2, 4, 8, 16, 20 e 24 horas. Nos testes para determinaÃÃo do PAA, apÃs cada um desses perÃodos seguiu-se uma inoculaÃÃo de 48 horas e para definiÃÃo do PAI, antes de cada perÃodo antecedeu-se um perÃodo de acesso de aquisiÃÃo fixo de 72 horas. Constatou-se que o PAA mÃnimo da mosca branca foi de apenas 0,25 hora, com o qual foram obtidas 6% de plantas infectadas. O percentual de plantas aumentou de 6 para 65% com a extensÃo do PAA de 0,25 para 24 horas. Com relaÃÃo ao perÃodo de acesso de inoculaÃÃo do vÃrus, foram registrados 18% de plantas infectadas com o PAI de 0,5 hora. O percentual elevou-se para 67% quando 24 horas de PAI foram concedidos. Valores isolados de 90 e 100% na transmissÃo viral, tambÃm foram observados. O tÃrmino do perÃodo latente do vÃrus no vetor ocorreu 16h apÃs a aquisiÃÃo do mesmo em planta infectada, considerando os 3% de infecÃÃo observados nas plantas inoculadas. Os dados obtidos indicam que a interaÃÃo vÃrus-vetor à estabelecida desde a fase inicial de desenvolvimento do inseto. Como parte do estudo dessa interaÃÃo, avaliou-se a presenÃa do begomovÃrus GO-ANPL em todas as fases de desenvolvimento do inseto vetor (ovo, 1 ao 4 Ãnstar e adulto) na planta infectada, em adultos com diferentes PAA, na progÃnie de fÃmeas virulÃferas e em adultos cujos estÃgios ninfais desenvolveram-se em tomateiro infectado. A tÃcnica PCR foi empregada para a detecÃÃo do GO-ANPL em mais de 2.500 espÃcimens testados. O vÃrus foi detectado em ninfas do 1 ao 4 Ãnstar que se alimentaram em plantas de tomate infectada, contudo, em ovos provenientes de avirulÃferas, os quais foram ovipositados em planta infectada e coletados apÃs sete dias, o vÃrus nÃo foi detectado. A transmissÃo à progÃnie foi constatada pela detecÃÃo do vÃrus em ovos, ninfas e adultos que se desenvolveram em planta nÃo hospedeira do vÃrus. A transmissÃo transestadial ocorreu com Ãndice elevado e, ao lado da transmissÃo à progÃnie, indica que a retenÃÃo do vÃrus à uma etapa importante da interaÃÃo vÃrusâvetor. A transmissÃo do vÃrus para mudas de tomate, a partir de adultos da progÃnie de fÃmeas virulÃferas, nÃo foi constatada. Contudo, transmissÃo para tomateiro em um percentual de 33% foi verificado nos casos em que a inoculaÃÃo das plantas foi realizada pelos adultos que retiveram o vÃrus da sua fase imatura (transestadial) / The whitefly-transmitted viruses from the family Geminiviridae, genus Begomovirus, have been reported as an economically important pathogen group that affect important crops in tropical and subtropical countries. Since the beginning of the 1980 decade, the occurrence of the whitefly associated to Begomovirus infection has drastically increased worldwide. In Brazil, these pathogens have been responsable for severe economical losses in tomato (Lycopersicon esculentum) orchards and the production has hampered since 1994. In this work, infected tomato plants showing symptoms, such as mosaic, intervein clearing, leaf curling and growth reduction were collected in tomato orchards in AnÃpolis, State of GoiÃs. The virus was identified as a member of the genus Begomovirus by PCR reaction, using specific primers to amplify fragments of A and B components of the virus DNA genome. The Chapter I of this thesis presents the results of the molecular characterization of the virus and the Chapter II shows the determination of its host range and the relationship with its natural vector Bemisia argentifolii. The virus isolate denoted GO-ANPL was cloned and partially sequenced. Part of the sequenced genome (2.180 nucleotides long) corresponded to the coat protein and Rep genes and comprised the entire intergenic region. Sequence comparison revealed that the GO-ANPL isolate is distantly related to the begomoviruses found in Asia, Europe and Africa, and it is related to other begomoviruses reported in Brazil. The virus isolate showed to be more closely related to viruses found in the State of Minas Gerais (TRMV isolate) and in the Federal District (isolate DF-Br2). The highest homology was observed with the isolate DF-Br2 and it may represent a new specie of the genus Begomovirus. In order to determine the virus host range, 46 plant species from nine different botanical families were mechanically and using the virus vector inoculated. The GO-ANPL isolate preferentially infected plants of the family Solanaceae as Nicotiana benthamiana, Datura stramonium and Nicandra physalodes. The number of infected plants was higher when they were inoculated by the virus vector, and the results were distinct from those obtained for other begomoviruses reported in Brazil. Viruses infections were all confirmed by dot blot hybridization using specific molecular probes to the virus. 4 To study virus/vector interaction, the acquisition access period (AAP), inoculation access period (IAP), and the latent period were determined transfering five whiteflies per plant and using tomato cv. Santa Clara as the host. For the AAP and IAP, nine different time periods were tested: 0.25, 0.5, 1, 2, 4, 8, 16, 20 and 24 h. The minimal AAP determined was 0.25 h, after which, 6% of the tested plants became infected. The number of infected plants increased to 65% with an AAP of24h.Afteran IAP of 0.5 h, 18% of the plants were infected and their number increased to 67% after an IAP of 24 hours. The latent period was considered to be 16 h, after which, 3% of the inoculated plants became infected. The results of AAP, IAP and latent period seem to indicate an early interaction between virus and vector starting at early stages of vector development. The presence of the GOANPL was determined in all stages of the vector (eggs, 1st to 4th instar and adults) in infected plants, in adults under different AAPs, in the progenies of viruliferous females, and in adults originated from nymphs developed from infected plants. More than 2.500 insects were tested by PCR to detect the GO-ANPL isolate. The virus was detected in nymphs from the 1st to 4th instar that had fed in infected plants and no virus was found in eggs from aviruliferous female that had been laid in infected plants. Transmission to the progenies was observed, since the virus was detected in all stages of insect development from eggs to adults. High level transmission was also observed in newly emerged adults that had acess to virus-infected plants as immatures. This fact, in addition to the transmission to the progenies, suggests that virus retention is an important part of virus/vector interaction. No transmission was observed from adults originated from viruliferous females. However, 33% of virus transmission was obtained when adults that retained virus from their early larval stages were employed
|
Page generated in 0.047 seconds