Cellular behaviour is regulated by a very precise system in which the complex interplay between various types of biomolecules plays a crucial role for the proper functioning of the system itself. Proteins interacting with DNA are in charged of its replication, packaging repair and recombination, among them, transcription factors regulate gene expression, but in turn they are part of a larger network of proteins interactions. In this thesis I addressed these aspects, in relation to a certain phenotype, in first instance by identifying common regulators of genes with similar expression signatures derived from high-throughput experiments, and then by computationally modelling the signal transduction by means of a message-passing algorithm. This allowed the identification of main regulators in the specific systems describing the infection process of Salmonella spp. in two different hosts: Arabidopsis thaliana and Homo sapiens. The same approach showed encouraging results in the pharmaco-dynamic study of drug-drug interactions. / El comportament cel•lular és regulat per un complex conjunt de relacions entre diferents tipus de biomol•lècules, que juga un paper fonamental per al correcte funcionament del propi sistema cel•lular. Diferents proteïnes s’encarreguen de la replicació, empaquetament, reparació i recombinació de l’ADN, i en regulen la seva expressió per mitjà de factors de transcripció. Aquests modulen la seva activitat mitjançant interaccions amb d’altres proteïnes, formant part d’una xarxa d’interaccions molt més extensa. En aquesta tesi m’interesso per aquests aspectes en relació a certs fenotips. Primer, identificant reguladors comuns de gens amb patrons d’expressió similars, obtinguts d’experiments d’alt rendiment; després, usant d’algorismes de transmissió del missatge per al modelat computacional de la transducció del senyal. Així he identificat reguladors principals en el procés d’infecció de Salmonella spp. en Arabidopsis thaliana i Homo sapiens. Una aproximació idèntica aporta resultats esperançadors en l’estudi de la farmacodinàmica de les interaccions entre drogues.

Hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol], a potent bioactive molecule mainly present in virgin olive oil and to a lower extent in wine, is also a by-product of dopamine metabolism. In a previous clinical trial designed to assess the effects of moderate wine intake it was found that hydroxytyrosol urinary recoveries were higher than the corresponding to the dose administered, suggesting an endogenous formation. The aim of the present work was to assess new mechanisms responsible for hydroxytyrosol generation by using an array of methodologies and studies ranging from in vitro assays to in vivo experiments in animal models and humans. The mechanisms identified as being involved in the generation of hydroxytyrosol were (1) CYP2A6/CYP2D6-catalyzed tyrosol-to-hydroxytyrosol biotransformation, (2) ethanol-induced increase in tyrosol bioavailability, and (3) alteration of dopamine oxidative metabolism due to ethanol. Considering these observations, it is postulated that hydroxytyrosol may contribute significantly to the health effects derived from moderate wine intake. / L’hidroxitirosol [2-(3,4-dihidroxiphenil)etanol], una potent molècula bioactiva present de forma rellevant a l’oli d’oliva verge i minoritàriament al vi, és també un producte del metabolisme de la dopamina. En un assaig clínic previ dissenyat per avaluar els efectes del consum moderat de vi es trobà que les recuperacions urinàries d’hidroxitirosol eren superiors a la dosi administrada, suggerint-ne una gènesi endògena. L’objectiu del present treball fou estudiar nous mecanismes responsables de la generació d’hidroxitirosol mitjançant diverses tècniques i estudis, des d’assajos in vitro a experiments in vivo en animals i humans. Els mecanismes involucrats a la generació d’hidroxitirosol són (1) la biotransformació de tirosol a hidroxitirosol catalitzada per CYP2A6/CYP2D6, (2) l’augment a la biodisponibilitat del tirosol induïda per l’etanol, i (3) l’alteració del metabolisme oxidatiu de la dopamina per l’etanol. Globalment, aquestes observacions donen a l’hidroxitirosol un paper clau en la comprensió dels efectes beneficiosos associats al consum moderat de vi.

Over the past years it has been described the important role of oxidative stress in the Metabolic Stress related manifestations, specifically in the obesity. It is well known that agents that reduce oxidative stress represent an important tool to reduce the obesity-induced complications such as hepatic steatosis. In this sense, flavonoids are described as antioxidant molecules, due to their scavenging properties. Moreover, omega-3 PUFAs are considered effective in the treatment and prevention of several chronic diseases. Therefore, the principal aim of this thesis was to study the antioxidant effects of flavonoids and n-3 PUFAs, as well as the possible additive effects of two compounds together. These studies were developed under several oxidative stress conditions (metabolic, chemical) both in vitro and in vivo. It has been demonstrated the importance of using nutritional supplements in different oxidative stress conditions. / Durant els últims anys s’ha descrit la importància que té l’estrès oxidatiu en les diferents patologies associades al Síndrome Metabòlic, especialment en l’obesitat. Així, agents que redueixen l’estrès oxidatiu son una important eina per reduir les complicacions relacionades amb l’obesitat, incloent la esteatosis hepàtica, definida com l’acumulació de greix al fetge. En aquest sentit, els polifenols, que presenten activitat antioxidant, i els àcids grassos poliinsaturats omega-3 que són considerats efectius en el tractament d’esteatosis hepàtica i en el desenvolupament de malalties cardiovasculars, són una bona eina per combatre aquest estrès oxidatiu. En aquesta tesi, s’ha estudiat en condicions d’estrès oxidatiu tant in vitro (hepatòcits) com in vivo (estat postprandial, estat d’obesitat induïda per la dieta o d’obesitat genètica) els efectes antioxidants dels flavonoides i/o omega-3 tant de manera individual com conjunta. S’ha demostrat la importància d’utilitzar suplements alimentaris en condicions metabòliques d’estrès oxidatiu.

L’objectiu d’aquesta tesis es desenvolupar una nova estratègia preventiva i terapèutica que requereixi les mínimes modificacions dels hàbits dietètics simultàniament efectiva en contra de l’obesitat i les seves malalties associades. Per aquest estudi s’han seleccionat compostos alimentaris presents en una dieta sana on el seus efectes beneficiosos han estat documentats tant en la obesitat com el risc de patir malalties cardiovasculars. S’inclou els polifenols com l’epigallocatechin gallate (EGCG) i les proantocyanidines i també els àcids grassos poliinsaturats omega 3 (PUFAs) o l’àcid docosahexaenoic (DHA). El treball esta enfocat en el funcionament mitocondrial del múscul, el teixit mes important de l’organisme en el control del balanç de nutrients. Els nostres resultats indiquen que hi ha una millora postprandial amb un increment de la capacitat d’oxidació dels àcids grassos millorant el perfil lipídic i la flexibilitat metabòlica. / El objetivo principal de esta tesis es desarrollar una nueva estrategia preventiva y terapéutica que requiera les mínimas modificaciones de los hábitos dietéticos simultáneamente efectiva en contra de la obesidad y sus enfermedades asociadas. Para este estudio se han seleccionado compuestos alimentarios presentes en una dieta sana dónde sus efectos beneficiosos han estado documentados tanto en la obesidad como en el riesgo de desarrollar enfermedades cardiovasculares. Se incluyen los polifenoles como el epigalocatequin galato (EGCG) y las proantocianidinas; también los ácidos grasos poliinsaturados omega 3 (PUFAs) y el acido docosahexaenoico (DHA). El trabajo está enfocado en el funcionamiento mitocondrial de musculo, el tejido más importante del organismo en el control del balance de nutrientes. Nuestros resultados indican que hay una mejora postprandial con un incremento de la capacidad de oxidación de los ácidos grasos mejorando el perfil lipídico y la flexibilidad metabólica. / The global goal of the present thesis is the development of a new preventative and therapeutic strategy that requires only minor modifications of dietary habits and simultaneously effective against obesity and its major associated diseases. For this study, we have selected food ingredients characteristic in healthy diets which has been documented beneficial effects on obesity or cardiovascular disease risk. These include plant polyphenols, epigallocatechin gallate (EGCG) and proanthocyanidins as well as the sea fish omega-3 polyunsaturated fatty acids (PUFAs) and docosahexaenoic acid (DHA). Our work is focused on the mitochondrial function of skeletal muscle, a major player in the body’s overall nutrient balance, and adipose tissue, which is the key regulator of energy homeostasis in the organism affected by obesity and related diseases. Our results indicate a postprandial improvement with an increase of the fatty acid oxidation capacity, improving lipid profile and metabolic flexibility.

The answer to whether or not, human pancreatic ribonuclease may have a diagnostic value as a serum marker of pancreatic disease, depend upon the specific detection of ribonucleases originated in pancreas amogn those coexisting in serum. From our point of view, two approaches would be necessary. On one hand the characterization of the carbohydrate component of the human pancreatic ribonuclease because glycosylation is organ-specific. On the other hand, obtain monoclonal antibodies in order to specifically recognize the secretory -typeribonuclease in serum. The purification system that has been established consists of two chromatographic steps using HPLC. The first, an exchange cromatography, allows for a partial purification of ribonuclease component. The second, a reversed-phase chromatography, resolves chromatographically the human pancreatic ribonuclease heterogeneity into different fractions / La resposta a si la ribonucleasa de pàncreas humà pot tenir algun valor diagnòstic com a marcador sèric de disfuncions pancreàtiques passa per la identificació de forma inequívoca de la ribonucleasa de pàncreas humà en sèrum. Segons el nostre enfoc, caldrien dues aproximacions, d’una banda la caracterització a nivell glucídic de la ribonucleasa de pàncreas humà, i de l’altra, el desenvolupament d’anticossos monoclonals dirigits a discriminar la ribonucleasa de tipus secretori d’entre els diferents tipus que coexisteixen en sèrum. En aquest sentit s’ha establert un sistema de purificació de la ribonucleasa pancreàtica humana que fos ràpid i repetitiu que respectés l’heterogeneïtat glucídica per a la seva posterior caracterització. El sistema de purificació establert a partir de pàncrees obtinguts a partir de donants sans d’òrgans consta de dues etapes cromatogràfiques per HPLC. La primera, una cromatografia de bescanvi catiònic, possibilita una purificació parcial, mentre la cromatografia de la fase inversa permet resoldre cromatogràficament l’heterogeneïtat de la ribonucleasa humana de pàncreas en diferents fraccions

Electrospinning is a manufacturing process that uses an electric field to produce fibers from a polymer solution. The accumulation of these fibers conform a three-dimensional fiber matrix or scaffold. Fibers can be prepared in a wide diameter range, namely from a micrometer to nanometer size. Furthermore, the fiber matrix or scaffold has a large surface per mass unit, a porous structure and mechanical properties influenced by the orientation of the fibers. The electrospinning technique is highly versatile and therefore a large number of polymers with different properties can be processed. However, a large number of variables can influence the characteristics of the resulting fibers, either because they are related to the polymer properties (e.g., solubility, molecular weight, etc.) or with the specific processing parameters (voltage, flow rate or distance tip-collector). Electrospun fiber matrices are attractive for biomedical applications as for example tissue engineering and drug delivery systems. In the last case, it is important the possibility to load the fibers with different drugs for their direct and localized administration into the human body. The goal of this Thesis is the study of different matrices constituted by electrospun micro- nanofibers and specifically four points have been considered. In the first one, polylactide electrospun scaffolds have been loaded with different molecules with antioxidant activity (i.e., vitamin B6 in pyridoxine and pyridoxal forms, p-coumaric acid and caffeic acid). The influence of these molecules on physical properties, morphology, in vitro release profiles and biocompatibility was determined. Furthermore, the application of these new materials for the inhibition of oxidative DNA damage caused by free radical initiators was demonstrated, and consequently, they appear appropriate candidates for purification of plasmidic or genomic DNA. In the second point, PLLA matrices loaded with two or three drugs were prepared in order to get a multifunctional activity. Thus, antioxidant, anti-inflammatory and antimicrobial molecules were considered in order to prevent chain oxidation processes in different biomolecules (proteins, DNA, etc.), avoid the subsequent local inflammation, and reduce the potential risk of microbial infection of wounds, respectively. These matrices are especially interesting due to the synergies and antagonisms that may occur during their simultaneous release. In the third point, the possibility of preparing biodegradable scaffolds from non electrospinable polymers has been considered. These polymers may have advantages like conductivity/electroactivity or bactericide activity. Hybrid scaffolds constituted by different ratios of polylactide as a biodegradable polymer and (poly(3-thiophene methyl acetate)) as electroactive polymer were evaluated. PLA nanofibers were also successfully loaded with polyhexamethylenebiguanide hydrochloride giving rise to 3D biodegradable scaffolds with a well proven antibacterial activity and a release that was highly dependent on the hydrophilicity of the medium. Finally electrospun scaffolds were obtained using a sacrificial polymer (e.g. poly(ethylene glycol) (PEG)) that could easily be subsequently removed by solubilization in aqueous media. Three approaches were evaluated: a) Preparation of scaffolds constituted by different ratios of PLA and PEG electrospun fibers, b) Preparation of scaffolds constituted by electrospun fibers with different PLA and PEG content; c) Preparation of scaffolds constituted by coaxial electrospun fibers with different core-shell polymer distributions. Cell colonization was in all cases favoured. The three procedures allowed preparing scaffolds with a differentiated drug release behavior. / La técnica de 'electrospinning' o electrohilado es un proceso de fabricación que utiliza un campo eléctrico para producir fibras a partir de disoluciones de polímeros. La acumulación de estas fibras conforma una matriz tri-dimensional o 'scaffold', y las fibras pueden ser preparadas en escala micro y nanométrica. Además, estas matrices o 'scaffold' se caracterizan por su gran superficie por unidad de masa, estructura porosa y propiedades mecánicas influenciadas por la orientación de las fibras. El 'electrospinning' es muy versátil y un gran número de polímeros con diferentes propiedades pueden ser procesados. Sin embargo, un gran número de variables pueden influir en las características de las fibras obtenidas, siendo variables propias del polímero (p.e., solubilidad, peso molecular, etc.) o relacionadas a los parámetros del proceso (voltaje, flujo, distancia colector-aguja). Estas matrices de fibras son atractivas para aplicaciones biomédicas como la ingeniería de tejidos y sistemas de liberación controlada de fármacos. En el último caso, es importante la carga de diferentes fármacos o drogas para su administración directa y localizada en el cuerpo humano. El objetivo de esta Tesis es el estudio de diferentes matrices constituidas por nano o microfibras electrohiladas. El desarrollo de este estudio se divide en cuatro bloques. En el primer bloque, matrices de fibras de poliláctico (PLA) fueron cargadas con diferentes moléculas con actividad antioxidante (vitamina B6 en sus formas de piridoxina y piridoxal, ácido p-cumárico y ácido cafeico). Se determinó la influencia de estas moléculas sobre las propiedades físicas, morfología, liberación in vitro y biocompatibilidad de dichas matrices. Además, se demostró la aplicación de estos nuevos materiales en la inhibición del daño oxidativo del ADN causado por iniciadores de radicales libres, y en consecuencia, estas matrices serían útiles para la purificación de ADN plasmídico o genómico. En el segundo bloque, las matrices de PLA fueron cargadas con dos o tres fármacos para obtener matrices multifuncionales en base a sus actividades. Con esta finalidad, moléculas con actividad antioxidante, anti-inflamatoria, y antimicrobiana fueron cargadas en las matrices para evitar los procesos de oxidación de diferentes biomoléculas (proteínas, ADN, etc.), evitar la inflamación local, y reducir el riesgo potencial de infección microbiana de las heridas, respectivamente. Estas matrices son especialmente interesantes debido a las sinergias y antagonismos que pueden ocurrir durante su liberación simultánea. En el tercer bloque, se prepararon matrices biodegradables a partir de polímeros no-electrohilables. Estos polímeros pueden presentar características particulares, como actividad bactericida, o actividad conductora/electroactividad. Matrices hibridas conformadas con diferentes ratios de PLA usado como polímero biodegradable y el poli(3-tiofeno metil acetato) como polímero electroactivo fueron preparadas y evaluadas. También se prepararon matrices de nanofibras de PLA cargadas con clorhidrato de polihexametilenbiguanida (PHMB) obteniéndose matrices biodegradables con actividad antibacteriana, y la liberación del PHMB fue altamente dependiente de la hidrófilicidad del medio. Finalmente, en el cuarto bloque, se prepararon matrices electrohiladas usando un polímero de sacrificio (polietilenglicol o PEG) que puede ser eliminado fácilmente por solubilización en medios acuosos. Tres preparaciones diferentes fueron evaluadas: a) Matrices constituidas por diferentes proporciones de PLA y PEG en las fibras, b) Matrices constituidas por fibras de PLA y fibras de PEG y, c) Matrices constituidas por fibras coaxiales con diferentes distribuciones de polímeros en el núcleo y la corteza de la fibra. La colonización celular en todas estas matrices fue mejorada. Estos tres procedimientos permitieron obtener matrices con diferentes comportamientos para la liberación de fármacos.

Epithelial to mesenchymal transition (EMT) is a process by which epithelial cells acquire a mesenchymal phenotype. It is characterized by the down-regulation of the adherens junction protein E-cadherin, and it is important during embryonic development. Snail1 expression is sufficient to trigger EMT in cultured cells and is found up-regulated in some cancers. Snail1 is stabilized both at mRNA and protein levels and in this project we analyzed the action of ubiquitin ligases affecting protein half-life. Apart from the already described β-Trcp1, that degrades Snail1 in a GSK-3β phosphorylation-dependent manner, we found the F-box proteins FBXL14 and FBXL5 as novel E3 ubiquitin ligases for Snail1. FBXL14 is a cytoplasmic ubiquitin ligase that is down-regulated in hypoxia through a transcriptional mechanism. FBXL5 is nuclear and modulates Snail1 binding to the DNA and nuclear ubiquitination. FBXL5 protein is destabilized after γ-irradiation, inducing high levels of Snail1. Together, these ligases keep a tight control of Snail1 cellular levels, maintaining them low in normal conditions. / La transició epiteli-mesènquima (per l’acrònim en anglès de: “epithelial to mesenchymal transition”, EMT) és un procés durant el qual cèl•lules epitelials adquireixen un fenotip mesenquimal. Està caracteritzat per la baixada de l’E-caderina, una proteïna de les unions adherents, i és important en el desenvolupament embrionari. L’expressió de Snail1 és suficient per desencadenar la EMT en cèl•lules en cultiu i s’ha trobat sobre-expressada en alguns càncers. Snail1 s’estabilitza tant a nivell de mRNA com de proteïna i en aquest projecte hem analitzat l’acció de les lligases d’ubiquitina que afecten els nivells de la proteïna. Apart de la ja descrita β-Trcp1, que degrada Snail1 de manera depenent a la fosforilació de Snail1 per GSK-3β, hem trobat les proteïnes F-box FBXL14 i FBXL5 con a noves lligases d’ubiquitina E3 que degraden Snail1. La FBXL14 és citoplasmàtica i els seus nivells disminueixen en hipòxia a través d’un mecanisme transcripcional. La FBXL5 és nuclear i modula tant la unió de Snail1 al DNA com la seva ubiquitinació nuclear. La proteïna FBXL5 es desestabilitza degut a la radiació gamma (γ) induint els nivells de Snail1.

Orientador: Elso Drigo Filho / Banca: Keizo Yukimitu / Banca: Nelson Augusto Alves / Banca: José Geraldo Nery / Banca: Marcelo de Freitas Lima / Resumo: As interações que ocorrem dentro de hemoproteínas são importantes para os sistemas biológicos e constituem termos de forte interesse para compreensão de sistemas vivos. Desta forma, é importante conhecer as interações vibracionais e eletrostáticas neste sistema. Neste trabalho foi feito um estudo sobre a interação entre o íon ferro(II) e o monóxido de carbono confinados em cavidades esféricas que mimetizam volumes de cavidades proteicas e um estudo do potencial de Morse confinado para o monóxido de carbono. Dessa forma, descrevemos como os autovalores de energia no estado fundamental para os potenciais da interação íon-dipolo e Morse se comportam no confinamento para um dos possíveis ligantes da mioglobina e da hemoglobina. Para o potencial de Morse desdobramos os cálculos para as seguintes moléculas diatômicas: nitrogênio, lítio e hidreto de sódio. Foi calculada, via Método Variacional, a energia para o sistema íon-dipolo confinado e potencial de Morse confinado. A função de onda teste sugerida foi inspirada na Mecânica Quântica Supersimétrica. Por fim, verificou-se como a energia de interação entre o íon e o dipolo se comporta com a mudança de permissividade do meio e como é o comportamento vibracional das moléculas de CO, N2, Li2 e NaH / Abstract: The interactions that occur within hemeproteins were important for biological systems and they were terms of strong interest for understanding living systems. ln this way, it is important to know the vibrational and electrostatic interactions in this system. ln this work a study is made on the interaction between iron ion and carbon monoxide confined in spherical cavities that mimic volumes of protein cavities and a study of the confined Morse potential for carbon monoxide. ln this way, we describe how the eigenvalues of energy in the ground state for the potentials of ion-dipole interaction and Morse behave in the confinement regime for one of the possible ligands of myoglobin and hemoglobin. For the Morse potential, we perform the calculations for the following diatomic molecules: nitrogen, lithium and sodium hydride. The energy for the confined ion-dipole system is calculated using the Variational Method. The suggested trial wavefunction is inspired by Supersymmetric Quantum Mechanics. Finally, it is verified how the energy of interaction between the ion and the dipole behaves in a change permittivity and how is the vibrational behavior of molecules of CO, N2, Li2 and NaH / Doutor

Submitted by Gizele Lima (gizele.lima@inpa.gov.br) on 2017-06-21T14:51:15Z No. of bitstreams: 2 Hallana_DISSERTAÇÃO_FINAL.pdf: 2393681 bytes, checksum: 019fc0eaef95f82f31a287ef01574305 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-21T14:51:15Z (GMT). No. of bitstreams: 2 Hallana_DISSERTAÇÃO_FINAL.pdf: 2393681 bytes, checksum: 019fc0eaef95f82f31a287ef01574305 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-12 / Fundação de Amparo à Pesquisa do Estado do Amazonas - FAPEAM / The transposable elements are known for their ability to move and integrate into the genome ofthe host organism randomly. They have two classifications: Class I is the retrotransposons' class that have as an intermediary transposition RNA, which from it is synthesized cDNA and this is inserted into the genome. The retrotransposons are classified in two ways, those with long terminal repeats, called retrotransposons LTRs and those who do not have this repetition is called retrotransposons not - LTRs. The retrotransposons not - LTRs can still be classified as LINEs (long interspersed elements) and SINES (short interspersed elements); Class II is composed of DNA transposons, which have their own DNA as an intermediary migrating or being directly copied and inserted into the genome. The Rex1 is retrotransposon not-LTR of Rex family and is found in several types of organisms. Several studies indicate that the Rex family retrotransposons have the capacity to respond to environmental stress. In this study we were made chromosome mapping and the absolute quantification Rex1 in Colossoma macropomum that have undergone environmental stress by water contamination with copper. The results corroborate the hypothesis that this retrotransposon has a response to that kind of stress in the muscle tissue of the Tambaqui. Found in chromosome mapping by fluorescence in situ hybridization, highest number oftags in animals which were subjected to stress and images were quantified in the FLIMA software, confirming that data through the highest fluorescence ratio. In Tambaqui absolute quantitation of muscle samples from the PCR Real - Time also found larger numbers ofcopies ofRex1 the samples were subjected to environmental stress copper. / Os elementos transponíveis são conhecidos pela capacidade de se mover e se integrar no genoma do organismo hospedeiro. Eles possuem duas classificações: a classe I é a classe dos retrotransposons que possuem como intermediário da transposição o RNA, a partir do qual é sintetizado o cDNA e este é inserido no genoma. Os retrotransposons são classificados de duas maneiras, os que possuem repetições terminais longas, chamados de retrotransposons LTRs, e os que não possuem essa repetição, sendo chamados de retrotransposons não – LTRs. Os retrotransposons não – LTRs ainda podem ser classificados como LINEs (elementos longos interespaçados) e SINEs (elementos curtos interespaçados); a classe II é composta por transposons de DNA, que possuem como intermediário o próprio DNA migrando diretamente ou sendo copiado e inserido no genoma. O Elemento Retrotransponível Rex1 é um retrotransposon não LTR da família Rex e é encontrado em vários tipos de organismos. Vários estudos apontam que os retrotransposons da família Rex possuem a capacidade de resposta ao estresse ambiental. Nesse estudo foram feitos o mapeamento cromossômico e a quantificação absoluta de Rex1 em Colossoma macropomum submetidos a estresse ambiental por contaminação da água com cobre. Os resultados encontrados corroboram a hipótese de que esse retrotransposon possua uma resposta a esse tipo de estresse no tecido muscular do Tambaqui. No mapeamento cromossômico encontramos, através da hibridização in situ por fluorescência, maior número de marcações em animais que foram submetidos ao estresse e as imagens foram quantificadas no software FLIMA, confirmando esse dado através do maior coeficiente de fluorescência. Na quantificação absoluta das amostras de músculo de Tambaqui a partir da PCR Real – Time, também encontramos maiores números de cópias de Rex1 nas amostras que foram submetidas ao estresse ambiental por cobre.

Made available in DSpace on 2014-06-11T19:31:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-30Bitstream added on 2014-06-13T20:41:05Z : No. of bitstreams: 1 apponi_lh_dr_araiq.pdf: 2630425 bytes, checksum: bd39235826e1f6c4a56f84fffab81fff (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A expressão gênica pode ser regulada em eucariotos em diversas etapas dometabolismo de mRNA, como transcrição, processamento, tradução e degradação. A estabilidade de mRNA é modulada por elementos presentes no transcrito e por proteínas ligantes de RNA associadas a esses elementos. Pub1 de S. cerevisiae é uma proteína citoplasmática capaz de estabilizar transcritos contendo elementos ricos em AU (ARE e ARE-like) ou elementos estabilizadores (STE). O presente trabalho identificou num rastreamento de duplo-híbrido a proteína Nab2 como ligante de Pub1. Nab2 é uma proteína nucleocitoplasmática essencial que regula o comprimento da cauda poli(A) e a exportação nuclear de mRNA. A interação entre Pub1 e Nab2 foi confirmada por co-purificação e ensaio de interação in vitro. Foi demonstrado também que essa interação é mediada pelo domínio de dedos de zinco presente na região C-terminal de Nab2. A análise da relação funcional entre essas duas proteínas revelou que Nab2, assim como Pub1, é capaz de modular a estabilidade de mRNA. A estabilidade do transcrito de RPS16B, mensageiro contendo sequência ARE-like e regulado por Pub1, é diminuída nos mutantes nab2- 1 e nab2-67. No entanto, a estabilidade do transcrito de GCN4, mensageiro contendo STE e também regulado por Pub1, não é afetada nos mesmos mutantes. Resultados semelhantes foram observados para outros transcritos contendo sequências ARE-like ou STE. Ainda, dados obtidos com um mutante da via NMD (?upf1) mostraram que esta via de decaimento não está envolvida com o mecanismo de estabilização de RPS16B mediada por Pub1 e Nab2. Uma análise mais profunda mostrou que a sequência ARE-like presente no mensageiro de RPS16B é necessária para a estabilização mediada por Nab2. A proteína Pub1 e seus domínios isolados foram produzidos e purificados, mas não foi possível a obtenção de cristais para... / Regulation of gene expression can occur at different levels of mRNA life cycle, including transcription, processing, translation and degradation. mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA-binding proteins. Poly(U)-binding protein 1 (Pub1) is a cytoplasmic S. cerevisiae mRNA binding protein that stabilizes transcripts containing AU-Rich Elements (ARE and ARE-like) or Stabilizer Elements (STE). In a yeast two-hybrid screen, we identified Nuclear poly(A)-binding protein 2 (Nab2) as a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by co-purification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific target mRNA transcripts. We find that the half-life of the RPS16B transcript, an ARE-likecontaining Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained with other ARE- and STE-containing Pub1 target transcripts. Additionally, results obtained with a mutant of the NMD pathway (?upf1) showed that this pathway is not involved in the mechanism of RPS16B stabilization mediated by Pub1 and Nab2. Further analysis reveals that the ARE-like sequence is necessary for Nab2- mediated transcript stabilization. Full-length Pub1 and isolated domains were produced and purified, however, it was not possible to obtain protein crystals for tertiary structure determination. Taken together, these results suggest that Nab2 acts together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events involved in mRNA biogenesis...(Complete abstract click electronic access below)