Field biomonitoring based on molecular biomarkers detects early warning signals of stress suffered by organisms exposed to contamination. The lower part of the Ebro River basin has a long pollution history related to the presence of a chlor-alkali plant adjacent to Flix. In addition, the Ebro River has been affected by the invasion of alien species that damage its natural ecosystem. In this thesis, Cyprinus carpio (common carp) an Dreissena polymorpha (zebra mussel) were used as a model species in laboratory and field studies intended to the development of a multimolecular biomarker approach to identify the most relevantly effects of pollutants in the field. Gene expression techniques were used to study specific physiological defensive mechanism. The application and improvement of a well-established markers in different aquatic species and following different approaches allowed to analyze modes of action and to outline effects of persistent organic and inorganic pollutants found in the field. The results showed that chronic exposure to mercury of common carp results in increased levels of metallothionein in kidney, in scales (albeit at lower extent), but not in liver, considered as the primary detoxification organ. The measure of gene expression in scales provides the possibility of a new non-lethal method of study. Studies of toxicant effects in zebra mussel revealed that this bivalve could be used as potential sentinel specie for freshwater monitoring. Analysis of its detoxicfication mechanisms, and particularly of its ABC membrane transporters in adult and the early life stages could help understanding the survival of this species in highly contaminated areas.

La transformación del mosto de uva en vino es un proceso microbiológico complejo aunque la fermentación alcohólica es llevada a cabo generalmente por cepas de Saccharomyces.El uso de inóculos de cepas preseleccionadas es frecuente en bodega dado que la cepa iniciadora se impone sobre las indígenas proporcionando ventajas encaminadas a una mayor producción y a una mejora de la calidad. El empleo de cultivos iniciadores hace necesario el establecimiento de criterios de selección de cepas con características deseables para su uso industrial. Además, el conocimiento en profundidad de la biología de estas levaduras permite el diseño de estrategias de mejora genética encaminadas a una mejora del proceso y/o de la calidad.C1. Caracterización fisiológica de cepas de Saccharomyces sp.: Estudio de la relación entre comportamiento fermentativo y resistencia a estrés.Se estudió el comportamiento fermentativo de diferentes cepas vínicas y se determinó su sensibilidad a diferentes condiciones de estrés demostrándose que existe una correlación estadísticamente significativa entre la capacidad de consumo de azúcares en condiciones de vinificación y la sensibilidad a dos condiciones de estrés, el oxidativo y el causado por adición de etanol.Los ensayos de resistencia a estrés pueden ser utilizados como criterios de selección de cepas con buen poder de fermentación. C2. Expresión de genes de respuesta a estrés durante la vinificación.Se analizó el perfil de expresión de genes de respuesta a estrés en diferentes condiciones de vinificación y en cepas con diferente comportamiento fermentativo. En cuanto a condiciones se ha visto que al inicio de la vinificación existe una influencia del pH del mosto y de la temperatura de incubación en la expresión de los genes analizados. En cuanto a expresión diferencial entre cepas se ha observado que las cepas con problemas fermentativos leves muestran en algunas fases de la vinificación y en algunos de los genes analizados niveles de expresión inferiores al resto de las cepas. En las cepas con problemas fermentativos graves el perfil de expresión es muy particular en varios de los genes mostrando algunos de ellos niveles máximos y mantenidos. A la vista de los resultados la correcta expresión de genes de respuesta a estrés parece importante para una adecuada adaptación de las levaduras a las condiciones de vinificación.C3. Expresión global en cepas con diferente comportamiento fermentativo durante la entrada en fase de crecimiento estacionaria.Se comparó la expresión génica (mediante micromatrices de DNA) y proteica (mediante electroforesis bidimensional) de una cepa con buen comportamiento fermentativo y otra cepa con leves problemas de vinificación. Así, se han podido determinar algunos factores moleculares que podrían explicar las diferencias fisiológicas entre estas dos cepas como aspectos relacionados con metabolismo de carbohidratos, con oxidación de compuestos orgánicos, con metabolismo del ergosterol y con respuesta a estrés. Además, se han observado diferencias de expresión de otros genes y proteínas que pueden ser relevantes para el uso industrial de estas levaduras.C4. Aplicaciones biotecnológicas. Se ensayó el comportamiento de las cepas en mostos naturales y partiendo de levadura seca activa demostrándose un comportamiento muy similar al que se había observado en vinificaciones con mosto sintético y con levaduras procedentes de cultivos líquidos.En segundo lugar se contruyeron cepas auxótrofas para uracilo de manera que se pueda estudiar el efecto de manipulaciones genéticas. Así, se puede ensayar la sobreexpresión de genes que mejoren la capacidad fermentativa en la cepa incapaz de completar el proceso de manera que no es necesario recurrir a condiciones limitantes de vinificación. / Must grape transformation into wine is a complex microbiological process although the alcoholic fermentation is mainly carried out by Saccharomyces strains. The use of preselected wine yeasts commercially available as active dry yeast is frequent since the initiator strain prevails on the natives providing advantages directed to a greater production and quality improvement. The use of this methodology makes necessary the establishment of criteria of wine yeast selection with desirable characteristics for its industrial use. In addition, a depth knowledge of the biology of these yeasts allows the design of genetic manipulation directed to an improvement of the process and/or a better quality of the final product. In this PHD project different wine yeast were characterized in order to obtain strains with different capabilities to conduct the vinification process. The resistance of these strains to different stress conditions was tested and we found a statistically significant correlation between the fermentative behaviour and the resistance to stress conditions. Later on we studied the molecular response of some of these yeasts analysing gene expression (RT-PCR quantitative and semi quantitative, micro arrays, macro arrays) and protein profiles (two-dimensional electrophoresis). With these studies it has been possible to determine some molecular factors that could explain the physiological differences between the strains like aspects related to carbohydrate metabolism, oxidation of organic compounds, metabolism of ergosterol and stress response. In addition, differences in the expression of other genes and proteins could be relevant for the industrial use of these strains. Finally, we tested the fermentative behaviour of these strains in conditions that try to mimic those which are found in the winery (natural must, active dry yeast) obtaining similar results to the previous experiments (synthetic must, liquid culture). Moreover we introduced an autotrophy in one of the strains with fermentative problems so it is possible to study the overexpression of genes which can improve the fermentative behaviour in non-limiting conditions.

Tesi realitzada a l'Institut d'Investigacions Biomèdiques de Barcelona (IIBB)- CSIC / 1) Estudio teórico de nucleobases modificadas. Las modificaciones químicas de ácidos nucleicos tienen una amplia variedad de aplicaciones tanto en clínica, utilizadas como agentes antisentido, así como en el estudio de las estructuras de los propios ácidos nucleicos, las interacciones proteína DNA o en la catálisis de ácidos nucleicos por poner algunos ejemplos. Actualmente se sintetizan cientos de análogos de nucleósidos estándar en laboratorios farmacéuticos, algunos de ellos como, por ejemplo, los análogos de la arabinosa de adenosina y citosina, son útiles como fármacos antivirales o anticancerígenos. La mayoría de los fármacos anticancerígenos actúan inhibiendo la síntesis de DNA de alguna manera si bien es cierto que tienen bastantes efectos secundarios. Sin embargo, la combinación de varios de estos fármacos reduce la resistencia y minimizan la toxicidad. Ejemplos de este tipo de análogos son los antimetabolitos que al ser estructuralmente muy similares a los nucleósidos estándar son capaces de interferir en la producción de los ácidos nucleicos inhibiendo las rutas de síntesis de ácidos nucleicos o incorporándose dentro de la estructura del DNA y el RNA. Una vez que se incorporan al DNA, estos compuestos provocan la terminación de la cadena durante la replicación del DNA y en consecuencia la muerte celular. Existen modificaciones del esqueleto azúcar-fosfato de los oligonucleótidos que mejoran la eficacia en la síntesis de siRNAs controlando la expresión génica en unos casos al bloquear estéricamente la actividad enzimática de las nucleasas, en otros inhibiendo la hibridación del mRNA con la hebra antisentido o incluso favoreciendo la formación de triplexes con secuencias específicas de DNA. Un ejemplo de ello son los fosforotioatos que poseen un átomo de oxígeno no enlazante sustituido por azufre en el enlace fosfodiéster. Éste tipo de modificaciones han demostrado ser resistentes tanto para exo- como endonucleasas aumentando la resistencia en cultivos celulares y en suero. Estudio de las propiedades tautoméricas y reconocimiento de las tiocetotiminas. Las bases sintéticas, una vez introducidas en el DNA, son capaces de inducir cambios conformaciones y alterar la estabilidad induciendo incluso mutaciones o modificando los patrones de reconocimiento. La forma tautomérica que suele suponerse para an álogos de nucleobases estándar es la forma ceto/amino. Sin embargo, la influencia del entorno puede en algunos casos favorecer la existencia de formas teóricamente minoritarias. En este estudio se describen las propiedades tautoméricas de la timina, y la 2- y 4-tiocetotiminas mediante métodos ab initio y la combinación de simulaciones de dinámica molecular y cálculos de energía libre. Los patrones de reconocimiento así como las estabilidades relativas de los tautómeros mayoritarios en diversos entornos fueron identificados. Los resultados teóricos fueron apoyados por los resultados experimentales aportados por el laboratorio del Prof. Dr. Ramón Eritja, y permiten describir las características tautoméricas y de reconocimiento de las tiocetotiminas. 2) Estudio de la influencia de la 6-selenoguanina en diferentes estructuras de DNA. Los análogos de purinas 6-mercaptopurina y 6-tioguanina se utilizan como agentes inhibidores del metabolismo de los ácidos nucleicos en diversos tipos de leucemias. En este trabajo presentamos una caracterización exhaustiva de la estructura y propiedades de interacción de la 6-selenoguanina, un isóstero de la guanina que introducido en el DNA puede afectar diferencialmente en estructuras de mayor orden del DNA. Mediante cálculos exactos de mecánica cuántica y la combinación de simulaciones de dinámica molecular con métodos de energía libre, hemos evaluado la influencia de la sustitución de la guanina estándar por la 6-selenoguanina en sistemas de doble, triple hebra de DNA así como en la estabilidad de estructuras G-cuádruplex. Finalmente, se aborda el estudio de las propiedades electrónicas de la 6-selenoguanina ya que se ha observado que ciertas modificaciones introducidas en el DNA podrían aumentar la capacidad conductimétrica de estos sistemas. 3) Funcionalización de los extremos 3' de oligos de DNA y RNA con derivados modificados con puentes N-etil-N y uso como inhibidor de 3'-exonucleasas. Existen ciertas limitaciones en el desarrollo de ácidos nucleicos modificados con aplicaciones clínicas y es que, la vulnerabilidad a la acción de nucleasas, tanto exo- como endonucleasas, limita la vida media de estos oligos en suero. En la actualidad, los fosforotioatos son las modificaciones que generan mayor resistencia aunque presentan la limitación de que un elevado número de los fosfatos del oligo han de ser modificados. En este trabajo presentamos una nueva modificación en el extremo 3’ de los oligos, en la que dos nucleósidos se unen por el lado de la base en lugar de por enlace fosfodiéster que es eliminado en el diseño. Simulaciones de dinámica molecular del complejo molecular con el fragmento Klenow de la polimerasa I de DNA de E. coli y el oligo modificado permiten demostrar la base del diseño racional del dímero modificado. Los estudios estructurales se complementaron con los experimentos realizados por la Dr. Montserrat Terrazas y Adele Alagia del grupo del Prof. Dr. Ramón Eritja. Éstos incluían estudios de digestión por 3’-exonucleasas (fragmento Klenow de la polimerasa I de DNA de E. coli y la fosfodiesterasa de veneno de serpiente) así como experimentos celulares de RNA de interferencia. 4) Estudio de la flexibilidad de ácidos nucleicos de doble cadena. El estudio de la flexibilidad de los ácidos nucleicos ha sido y sigue siendo un campo actual en efervescencia gracias a la desarrollo de las técnicas de moléculas individuales y a los métodos computacionales como la dinámica molecular. En este estudio se aborda la influencia de la secuencia en la estructura, estabilidad y flexibilidad de moléculas de RNA de doble cadena mediante simulaciones de dinámica molecular. Otro de los objetivos del estudio era comparar los dos campos de fuerza que son mayoritariamente utilizados en el campo de los ácidos nucleicos, AMBER (parm99bsc0) y CHARMM (CHARMM27). Los resultados obtenidos demuestran que la flexibilidad es dependiente de la secuencia pudiendo agrupar los diferentes dinucleótidos en su mayor o menor capacidad para ser deformados. Medidas experimentales como la longitud de la persistencia o la torsión de las dobles hélices de RNA son parámetros moleculares globales que definen la flexibilidad en una escala mayor. Mediante métodos de dinámica esencial, se calcularon los promedios de las entropías intramoleculares de las cuatro secuencias consideradas así como las estimaciones teóricas de la longitud de la persistencia, de la torsión helicoidal y del estiramiento molecular. Además, los resultados del estudio aportan más datos acerca de la diferente flexibilidad que existe entre el DNA y el RNA de doble cadena. / In this thesis quantum mechanics and classical mechanics techniques have been used to study the tautomeric properties of modified nucleobases with potential application to antisense and biotechnology fields. On the other hand, the study of the mechanical properties of double stranded DNA and RNA (dsDNA versus dsRNA) and their sequence dependency have been addressed. In practice, the work has been divided into the following sections: 1.- Theoretical study of sulfur modified thymines. The tautomeric properties of thymine and two thioketoderivatives, 2- and 4-thioketothymines, have been studied by means of accurate ab initio calculations and molecular dynamics simulations coupled with free energy calculations. Previous results suggested that these modifications could stabilize DNA structure at least as good as the natural thymine leading to the fidelity consequences during the replication. Indeed, those results suggested that some minor tautomeric forms could be present in the DNA double stranded environment which would explain the higher stability of certain mismatches. In the light of these results, we explored the impact of thioketothymines by means of both theoretical and experimental studies. 2.- Theoretical study of seleno modified guanine. We explore the chemical properties of 6-selenoguanine (6SeG) and the influence in duplex, triplex and G-quadruplex structures by means of high-level quantum mechanics calculations and free energy calculations combined with molecular dynamics simulations. 6-Selenoguanine, like other related antimetabolites like thiopurines, has been longtime used against several cancers like lymphomas or hepatomas. Moreover, their applications have been extended to solving X-ray nucleic acids structures (6SeG can be incorporated to these molecules and display anomalous dispersion) and, from the present work we suggest that 6SeG can also exhibit remarkable conductor properties in DNA structures. 3.- Inhibition of 3’-exonucleases with dimeric N-ethyl-N modified nucleosides. Development of synthetic oligonucleotides (ONs) has received much attention in the past years due to their ability to silence the expression of undesired overexpressed genes. Among established therapies, siRNAs have been widely studied due to their high potency and sequence-specificity. However, the application of oligonucleotides in vivo faces important limitations. For example, their high vulnerability to degradation by serum nucleases. Much effort has been made to overcome these limitations but, in many cases, modifications that increase nuclease stability, cause negative effects on RNAi activity. We are interested in creating chemical modifications able to increase nuclease stability without disrupting RNAi activity. In particular, in this work, we explored 3’-terminal modifications by replacing standard nucleobases by N-ethyl-N-coupled nucleosides. We focused special attention on 3’-exonucleases because they are the predominant nuclease activity present in serum. By means of molecular dynamics simulations and in vitro experiments, modified siRNAs resistant to nuclease digestion were evaluated against Bcl-2 in vitro, an antiapoptotic gene which is overexpressed in several cancer processes. 4.- Flexibility study of dsRNA molecules. Despite their chemical similarity, DNA and RNA duplex structures play very different biological roles. While double stranded DNA can display a large variety of structures close to the B-form with very dynamic sugar-phosphate conformations, RNA double stranded has been traditionally considered rigid. However, presented results from CHARMM27 MD simulations that pointed in the opposing direction, stating that RNA2 is more flexible than DNA2. To shed some light on this topic, we study the flexibility of double stranded RNA molecules by means of molecular dynamics simulations on four different 18-mer sequences. AMBER and CHARMM nucleic acid force fields were used to analyze the sequence dependent elastic properties and the results were ultimately compared with the obtained for DNA duplex, were both force fields produced comparable results.

Cell migration is a fundamental element in a variety of physiological and pathological processes. Alteration of its regulatory mechanisms leads to loss of adhesion and increased motility, critical steps in the initial stages of metastasis. Consequently, cell migration has become the focus of intensive experimental and theoretical studies; however the understanding many of its mechanisms remains elusive. Cell migration is the result of a periodic sequence of protrusion, adhesion remodeling and contraction stages that leads to directed movement towards external stimuli. The spatio-temporal coordination of these processes depends on the activation of the signaling networks that regulate them at specific subcellular locations. Particularly, the family of small RhoGTPases plays a central role in regulating cell polarization, the formation of adhesion sites and the generation of the forces that drive motion. Theoretical models based on an independent description of these processes have a limited capacity to predict cellular behavior observed in vitro, since their functionality depends on the cross-regulation between their signaling pathways. This thesis presents a model of cell migration that integrates a description of force generation and cell deformation, adhesion site dynamics and RhoGTPases activation. The cell is modeled as a viscoelastic body capable of developing traction and protrusion forces. The forces are determined by the activation level of the RhoGTPases, whose distribution in the cell is described by a set of reaction-diffusion equations. Adhesion sites are modeled as punctual clusters of transmembrane receptors that dynamically bind and unbind the extracellular matrix depending on the force transimtted to them and the distance with ligands coating the substrate. On the theoretical level, the major findings relate the topology of a Crosstalk Scheme and the properties inherited by the associated reaction network as a gradient sensing and regulatory system: reversible polarization, adaptation to uniform stimulus, multi-stimuli response and amplification. Models formulated according to these principles remain functional against the biological diversity associated to different cell types and match the observed behavior in Chemotaxis essays: the capacity of cells to detect shallow gradients, polarize without featuring Turing patterns of activation, and switch the direction of migration after the stimulus source is changed. The biological implications challenge a long held view on the mechanisms of RhoGTPase crosstalk and suggests that the role of GDIs, GEFs and GAPs has to be revised, as supported by recent experimental evidence. In addition, the model recapitulates a continuous transition from the tear-like shape adopted by neutrophiles to the fan-like shape of keratocytes during migration by varying the magnitudes of protrusion and contraction forces or, alternatively, the strength of RhoGTPase Crosstalk. The second mechanism represents a novel explanation of the different morphologies observed in migrating cells. On cell mechanosensing, a new hypothesis is proposed to explain how cells sense the mechanical properties of the ECM. The hypothesis provides a unifying explanation to apparently conflicting observations on force development and growth in real time at cell Focal adhesions, previously attributed to differences in experimental set-ups or cell types studied. An interpretation for the observed relationships between polarization time, migration speed, mechano-sensing limits and substrate rigidity follows from this hypothesis. Further, the theory directly suggests the currently unknown mechanisms that could explain the universal preference of cells (bar neurons) to migrate along stiffness gradients, and for the first time, a plausible biological function for the existence of this phenomenon. It is known as Durotaxis, and its abnormal regulation has been associated to the malignant behaviour of cancer cells.

L'objectiu principal d'aquesta tesi ha estat l'estudi de l'efecte de les condicions ambientals d'il.luminació en què un animal ha crescut sobre la manifestació dels seus ritmes circadiaris, que són els mecanismes que els éssers vius utilitzen per a adaptar-se a l'alternància entre el dia i la nit.El treball s'ha estructurat en dues parts: la primera on s'ha estudiat, en animals sans, els efectes de les condicions ambientals rebudes durant les primeres setmanes de vida en el desenvolupament i funcionalitat del sistema circadiari, i una segona on s'ha treballat amb rates hipertenses de la soca TGR(mREN2)27, amb la mateixa finalitat.

Η μελέτη αυτή αφορά το σχεδιασμό των μιγμάτων πρωτεΐνης/πολυηλεκτρολύτη και τις αλληλεπιδράσεις τους σε φυσιολογικές συνθήκες. Η παρακάτω έρευνα περιλαμβάνει σύμπλοκα αλβουμίνης ορού βοοειδούς (BSA) με ένα σύνολο κατιονικών ομοπολυμερών και συμπολυμερών πολύ (3-μεθακρυλαμιδο-προπυλο- τριμεθυλαμμωνιο-χλωριδιο) -co- (Ν, Ν-διμεθυλακρυλαμίδιο), με διαφορετικό μοριακό βάρος και σύσταση σε ομάδες πολυ (Ν, Ν- διμεθυλακρυλαμιδίου) (PDMAM). Μετρήσεις UV-VIS φασματομετρίας, η σκέδαση φωτός και ζ-δυναμικό συμμετείχαν στο χαρακτηρισμό των συσσωματωμάτων ως μία συνάρτηση της αναλογίας φορτίου r +/- , του pΗ και της ιοντικής ισχύος. Τα αποτελέσματα έδειξαν το σχηματισμό αντίθετων φορτισμένων συμπλόκων, σε pΗ υψηλότερο από 4.9, το ισοηλεκτρικό σημείο της BSA. Τέτοια σωματίδια βρέθηκαν να εξαρτώνται από την πυκνότητα φορτίου της επιφάνειας της πρωτεΐνης η οποία επηρεάζει σημαντικά τις ηλεκτροστατικές αλληλεπιδράσεις. Ως αποτέλεσμα, για ένα ομοπολυμερές PMAPTAC ο σχηματισμός αδιάλυτων συμπλοκών ευνοείται για υψηλές τιμές pΗ, όπου η πυκνότητα επιφανειακού φορτίου της πρωτεΐνης είναι μέγιστη. Αντίθετα, αύξηση της ιοντικής ισχύος οδηγεί σε καταστολή των απώσεων μακράς εμβέλειας μεταξύ των αρνητικά φορτισμένων περιοχών του πολυηλεκτρολύτη και της πρωτεΐνης, εξαιτίας τοπικών ηλεκτροστατικών επιδράσεων. Οι διαφορές στο σχηματισμό συμπλόκου επιβεβαιώθηκαν με σύγκριση δύο PMAPTAC ομοπολυμερών με διαφορετικά μοριακά βάρη σε σταθερό pΗ. Αυτά τα συσσωματώματα έδειξαν διαφορές στο μέγεθος και τις επιφανειακές ιδιότητες οδηγούμενες από την πυκνότητα φορτίου του πολυηλεκτρολύτη. Έτσι, τα μεγαλύτερα σωματίδια ελήφθησαν από το πολυκατιόν με υψηλότερο μοριακό βάρος σε pΗ 7,40. Ακόμα, ο ρυθμός κατανάλωσης της BSA για το σχηματισμό συμπλόκου με τον πολυηλεκτρολύτη ήταν παρόμοιος για όλες τις περιπτώσεις διαφορετικού pH δίνοντας έτσι το γεγονός ότι η καταβύθιση της BSA είναι ανεξάρτητη του pH. Η προσθήκη ουδέτερα υδρόφιλου PDMAM επεκτείνει το πιθανό μηχανισμό συμπλοκοποίησης. Με τη σύγκριση μιας σειράς από συμπολυμερή που φέρουν διαφορετικό αριθμό των υδρόφιλων ομάδων DMAM, μόνο διαλυτά σύμπλοκα πολυηλεκτρολύτη / πρωτεΐνης σχηματίζονται με υδροδυναμικά μεγέθη <50 nm. Η συμπεριφορά αυτή έχει αποδοθεί στην παρουσία των υδρόφιλων μονάδων DMAM που απλά δρα ως προστατευτικό εξωτερικό κέλυφος των συσσωματωμάτων εμποδίζοντας περαιτέρω συσσωμάτωση. Συνεπώς, από τα σύμπλοκα που σχηματίζονται από το συμπολυμερές με την διαφορετική περιεκτικότητα DMAM, ισχυρότερη προτίμηση ένωσης με την πρωτεΐνη έδειξε το συμπολυμερές με τη χαμηλότερη περιεκτικότητα DMAM. Αυτό δείχνει τη σημασία των υδρόφιλων ομάδων οι οποίες παρεμποδίζουν στερεοχημικά την πρόσβαση ή έκθεση των ιόντων στην επιφάνεια του συμπλόκου. Ακόμα, το υδρόφιλο εξωτερικό κέλυφος είναι είτε μικρότερο, είτε λιγότερο πυκνό, γεγονός που επιτρέπει την αναδιάταξη των σωματιδίων έτσι ώστε περισσότερα φορτία να είναι παρών στην επιφάνεια του 4 συσσωματώματος που μπορεί περαιτέρω να αλληλεπιδρούν με το κατιονικό πολυηλεκτρολύτη. Έτσι, το σύστημα που παρουσιάζεται εδώ δίνει μία εις βάθος μελέτη των πιθανών μηχανισμών των αλληλεπιδράσεων πολυηλεκτρολύτη με πρωτεΐνες. Επιπλέον, ο σχηματισμός συμπλοκών μικρής κλίμακας σταθερών σε υδατικό διάλυμα επιτεύχθηκε με ρύθμιση παραμέτρων, όπως το μοριακό βάρος, η αναλογία φορτίου και της ισορροπίας υδρόφιλου / υδρόφοβου. Επιπρόσθετα, τα συστήματα ήταν σε συμφωνία με περιβαλλοντικές συνθήκες όπως το pΗ και την ιοντική ισχύ. Τέτοια σωματίδια μπορούν να χρησιμοποιηθούν για παροχή φαρμάκου με καλή σταθερότητα σε φυσιολογικές συνθήκες που μπορεί να οδηγηθεί μέσω της ροής του αίματος. Επιπλέον, η ευαισθησία στο pΗ των συστημάτων αυτών τα καθιστά εξαιρετικούς υποψηφίους για θεραπεία του καρκίνου, όπου οι διαφορές στο pH μεταξύ καρκινικών και φυσιολογικών κυττάρων μπορούν να δώσουν ιδέες για απελευθέρωση στοχευόμενου φαρμάκου. / This study concerns the design of protein/polyelectrolyte mixtures for complexion at physiological conditions. The concept herein involves bovine serum albumin (BSA) complexes with a set of cationic homopolymers and copolymers of poly (MAPTAC)-co -poly (N, N-dimethylacrylamide), with different molecular weight and composition in poly (N, N-dimethylacrylamide) (PDMAM) groups. UVVIS spectrometry, Light Scattering measurements and zeta-potential were involved in the characterization of the aggregates as a function of charge ratio r +/- , pH and ionic strength. The results revealed the formation of oppositely charged complexes, at pH higher than 4.9, the isoelectric point of BSA. Such particles were found dependent on the charge density of the protein surface which strongly influences the electrostatic interactions. As a result, for a PMAPTAC homopolymer the formation of insoluble complexes is favored for high pH values, where the surface charge density of the protein is maximum. Contrarily, enhancement of ionic strength conditions had led to suppression of long-range repulsion between polycation and protein negative domains due to electrostatic screening effects. The differences in the complexation were confirmed by comparing two PMAPTAC homopolymers with different molecular weights at constant pH. These aggregates showed differences in size and surface properties dictated by the charge density of the polyelectrolyte. Thus, larger particles were obtained from the polycation with higher molecular weight at pH 7.4. Interestingly, the rate of BSA consumption for polyelectrolyte formation was identical for all the occasions of pH leading to the conclusion that the consumption of BSA is independent of pH. Addition of neutral hydrophilic PDMAM extended the possible complexation mechanism. By comparing a set of copolymers bearing different content of hydrophilic DMAM groups, only soluble polyelectrolyte/ protein complexes were formed with hydrodynamic sizes < 50 nm. This behavior has been ascribed to the presence of the hydrophilic DMAM units that acts as a protective hydrated outer shell of the aggregates refraining further aggregation. Accordingly, the complexes formed from the copolymer with the different DMAM content have shown 2 stronger protein affinity for the copolymer with the lowest DMAM content. This illustrates the importance of hydrophilic groups which sterically hinder the access and/or exposure of ions to the surface of the complex. Thus the hydrophilic outer shell is either shorter or less dense, fact that allows rearrangement of the particles so that more charges are present on the surface of the aggregate that can further interact with the cationic polyelectrolyte. Thus, the system presented herein represents an in depth study of possible mechanisms of polyelectrolyte interactions with proteins. Furthermore, the formation of nano-scaled complexes stable in aqueous solution was achieved by tuning molecular parameters such as molecular weight, charge ratio and hydrophilic/hydrophobic balance. Additionally, the systems had presented responsiveness to environmental stimuli such as pH and ionic strength. Such particles can be used for drug delivery with good stability at physiological conditions which can lead to long circulation in the blood stream. Moreover, the pH sensitivity of these systems makes them excellent aspirants for cancer therapy where differences in tumor and normal cell pH is a concept for targeted drug delivery.

Controlled release systems have become an innovative technique to treat diseases like cancer by the targeted delivery to individual cells and tissues. There is an urgent need to achieve efficacious and safe delivery with minimal nonspecific uptake by healthy tissues. Among the polymer-based nanoparticulate systems for drug delivery, nanoparticles (NPs) have represented a promising opportunity as delivery system due to their degradation in water-soluble compounds that enter the normal metabolic pathways of the organism and their capacity to modify pharmacokinetics and the drug tissue distribution profile. An engineered and versatile targeted nano-platform for the delivery of paclitaxel (PTX) across the blood brain barrier (BBB) with the aim to improve its therapeutic effect on human glioma cells has been developed. A novel biodegradable polymer has been synthetized and custom tailored NPs have been obtained. The method allows to modify the targeted drug delivery for efficiently transport and release of active drug molecule across the BBB. Aiming a dual targeting strategy, functionalization with ligands known to be efficiently transported across BBB by a membrane receptor that also is over-expressed on human glioma cells has been employed to shuttle PTX from blood to brain and then target glioma cells. In vivo properties of the NPs have been explored to assess their biological profile and since the pressing need for careful evaluation, new strategies for NPs radiolabeling with the aim to investigate their in vivo fate, specifically stability in biological environments (stealthiness), biodistribution and pharmacokinetic, have been adopted. / Los sistemas de liberación controlada de medicamentos, mediante la administración dirigida individualmente a células y tejidos, se han convertido en una técnica innovadora para tratar enfermedades como el cáncer. Existe una necesidad urgente para lograr una liberación eficaz y segura que incluya una mínima absorción no específica para los tejidos sanos. Entre los sistemas nanopartículados a base de polímeros para la administración de fármacos, las nanopartículas (NPs) han representado una oportunidad prometedora como sistema de suministro. Entre sus ventajas se puede destacar su perfil de degradación en compuestos hidrosolubles y no tóxicos, que se eliminan siguiendo las vías metabólicas normales del organismo. Por otro lado, presentan una elevada capacidad de modificar la farmacocinética y el perfil de distribución del medicamento en los tejidos. En esta tesis se ha desarrollado una nano-plataforma específica y versátil para la liberación de paclitaxel (PTX) a través de la barrera hematoencefálica (BHE) con el objetivo de mejorar su efecto terapéutico sobre las células de glioma humano. Se ha sintetizado un nuevo polímero biodegradable gracias al cual se han obtenido NPs personalizadas a medida. El método permite modificar el tipo administración dirigida de los fármacos para conseguir un transporte y una liberación de las moléculas de principio activo eficiente y segura. Se ha desarrollado el objetivo de seguir una estrategia de selección dual que consiste en transportar el PTX desde la sangre hasta el cerebro y luego dirigirse a las células de glioma. Para ello se ha empleado la funcionalización con marcadores capaces de atravesar eficientemente la BHE a través de un receptor de membrana que también está sobre-expresado en las células de glioma humano. Para evaluar el perfil biológico de las NPs se han explorado sus propiedades in vivo y dada la urgente necesidad de una evaluación fiable, se han adoptado nuevas estrategias para radiomarcar NPs con el objetivo de investigar su destino in vivo, la estabilidad en entornos biológicos, la biodistribución y la farmacocinética. / Els sistemes d'alliberament controlat de medicaments, mitjançant l'administració dirigida individualment a cèl•lules i teixits, s'han convertit en una tècnica innovadora per tractar malalties com el càncer. Hi ha una necessitat urgent per aconseguir un alliberament eficaç i segura que inclogui una mínima absorció no específica per als teixits sans. Entre els sistemes nanoparticulats a base de polímers per a l'administració de fàrmacs, les nanopartícules (NPs) han representat una oportunitat prometedora com a sistema de subministrament. Entre els seus avantatges es pot destacar el seu perfil de degradació en en compostos hidrosolubles i no tòxics, que s'eliminen seguint les vies metabòliques normals de l'organisme. D'altra banda, presenten una elevada capacitat de modificar la farmacocinètica i el perfil de distribució del medicament en els teixits. En aquesta tesi s'ha desenvolupat una nano‐plataforma específica i versàtil per a l'alliberament de paclitaxel (PTX) a través de la barrera hematoencefàlica (BHE) amb l'objectiu de millorar el seu efecte terapèutic sobre les cèl•lules de glioma humà. S'ha sintetitzat un nou polímer biodegradable gràcies al qual s'han obtingut NPs personalitzades a mida. El mètode permet modificar el tipus administració dirigida dels fàrmacs per aconseguir un transport i un alliberament de les molècules de principi actiu eficient i segura. S'ha desenvolupat l'objectiu de seguir una estratègia de selecció dual que consisteix a transportar el PTX des de la sang fins al cervell i després dirigir‐se a les cèl•lules de glioma. Per a això s'ha emprat la funcionalització amb marcadors capaços de travessar eficientment la BHE a través d'un receptor de membrana que també està sobre-expressat en les cèl•lules de glioma humà. Per avaluar el perfil biològic de les NPs s'han explorat les seves propietats in vivo i donada la urgent necessitat d'una avaluació fiable, s'han adoptat noves estratègies per radiomarcar NPs amb l'objectiu d'investigar la seva destinació in vivo, l'estabilitat en entorns biològics, la biodistribució i la farmacocinètica.

Submitted by Elizabete Silva (elizabete.silva@ufes.br) on 2014-12-05T16:38:39Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao. Jesus Fernando.pdf: 7436239 bytes, checksum: f1de263a3086e7049699b2f892aa6825 (MD5) / Approved for entry into archive by Elizabete Silva (elizabete.silva@ufes.br) on 2014-12-05T16:46:14Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao. Jesus Fernando.pdf: 7436239 bytes, checksum: f1de263a3086e7049699b2f892aa6825 (MD5) / Made available in DSpace on 2014-12-05T16:46:14Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertacao. Jesus Fernando.pdf: 7436239 bytes, checksum: f1de263a3086e7049699b2f892aa6825 (MD5) Previous issue date: 2014 / A intensa atividade humana devasta grandes extensões de florestas nativas, seja para expansão da agricultura seja para suprir a crescente demanda do mercado por madeira, de uma forma ou de outra os pequenos fragmentos florestais remanescentes sofrem constantes pressões antrópicas, contudo, o uso de espécies arbóreas adaptadas pode contribuir para a proteção desses recursos naturais. Buscou-se neste estudo avaliar os padrões fenológicos de 109 espécies florestais arbóreas pertencentes a 37 famílias e 82 gêneros, dentre nativas e exóticas, com nove anos de idade, para diferenciar as mais adaptadas e com potencial de serem utilizadas em programas de reflorestamento. O estudo foi realizado na Fazenda Experimental do Incaper, em Jucuruaba, município de Viana-ES, (UTM E-345524, N- 7741039). Foram realizadas análises químicas do solo na área plantada e os dados climatológicos obtidos na estação meteorológica de Viana. O estudo baseou-se na observação do número de plantas sobreviventes de cada espécie e da avaliação do seu crescimento. Foram realizadas observações das fenofases de brotação, senescência de folhas, floração e frutificação. As avaliações fenológicas foram realizadas em intervalos mensais, no período de novembro de 2012 a outubro de 2013. Realizou-se a medição da altura das árvores, diâmetro à altura do peito (DAP), índice de enfolhamento, taxa de sobrevivência e cálculo do ICC (Índice Combinado de Crescimento), bem como a determinação das espécies mais adaptadas. Das 109 espécies estudadas, 64,22% apresentaram adaptação funcional e estrutural às condições de solo e clima da região experimental, 42,22 % floresceram e frutificaram e 90% apresentaram senescência e brotação acompanhando a sazonalidade climática. Vinte e nove espécies apresentaram ICC maior do que o ICC médio. A maioria das espécies destacou-se como alternativa para recuperação da cobertura vegetal local, com destaque para Inga uruguensis e Schizolobium amazonicum. As análises de fluorescência da clorofila revelaram que o aparato fotossintético da Schizolobium amazonicum foi capaz de protegê-la da fotoinibição e promover boa conversão da energia luminosa em fotoquímica. / The intense human activity devastates large native forests areas, either for agriculture expansion or to supply the growing wood market; anyway, the small forest fragments remaining suffer constant anthropic pressure. However, the use of adapted tree species may contribute to the natural resources protection. This present study aimed the evaluation of phenological patterns from 109 forest tree species, belonging to 37 families and 82 genders, between native and exotic, with 9 years old, to differentiate the most adapted and with potential to be used in reforesting programs. The search was conducted at Incaper’s Experimental Farm, in Jucuruaba, Viana-ES city, (UTM E-345524, N-7741039). It was performed soil chemical analysis of the planted area and the weather data was obtained in Viana’s meteorological station. The study was based on the observation of the number of surviving plants from each specie and its growth evaluation. Observations of the budding phenophases, leaves senescence, flowering and fruiting were performed. Phenological evaluations were developed monthly, from November 2012 to October 2013. It were collected data from the measurement of tree height, diameter at breast height (DBH), index of foliage, survival rate and calculating the CGI (Combined Growth Index), as well as the determination of the most appropriate species. From the 109 species studied, 64.22% showed functional and structural adaptation to soil and climate conditions of the experimental region; 42.22% flowered and fruited; and 90% showed senescence and budding following seasonality. Twenty-nine species showed a GIC higher than the average. Most species stood out as an alternative to local vegetation recovery, especially Inga uruguensis and Schizolobium amazonicum. The chlorophyll fluorescence analysis revealed that the photosynthetic apparatus of Schizolobium amazonicum was able to protect it from photoinhibition and promote good conversion of light energy into photochemical.

Submitted by Elizabete Silva (elizabete.silva@ufes.br) on 2014-12-15T18:00:16Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO LARISSA MARIA FURLAN.pdf: 1744992 bytes, checksum: 71ef1054ac66b9ee2f174b0fd2b645d3 (MD5) / Approved for entry into archive by Elizabete Silva (elizabete.silva@ufes.br) on 2014-12-16T19:19:27Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO LARISSA MARIA FURLAN.pdf: 1744992 bytes, checksum: 71ef1054ac66b9ee2f174b0fd2b645d3 (MD5) / Made available in DSpace on 2014-12-16T19:19:27Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) DISSERTAÇÃO LARISSA MARIA FURLAN.pdf: 1744992 bytes, checksum: 71ef1054ac66b9ee2f174b0fd2b645d3 (MD5) Previous issue date: 2013 / A luminosidade desponta como um dos fatores abióticos de maior importância no estabelecimento das plantas, podendo ocasionar alterações a nível fisiológico e anatômico, interferindo diretamente no desenvolvimento das mesmas e consequentemente, sua produtividade. O cacaueiro (Theobroma cacao – Malvaceae) possui grande interesse econômico devido à utilização de suas sementes para produção de manteiga de cacau e chocolate. Neste sentido, o objetivo deste trabalho foi comparar características fisiológicas e anatômicas de dois genótipos de cacaueiro (PH 16 e IPIRANGA 01), submetidos a diferentes condições de radiação solar de modo a inferir a respeito das condições de luminosidade mais favoráveis ao desenvolvimento dos mesmos. Foram realizadas análises de crescimento, teor de pigmentos, trocas gasosas e anatômicas caulinares e foliares. O delineamento experimental foi em Blocos casualizados (DBC), com 4 repetições em arranjo fatorial 2 x 5, constituído de dois genótipos e 5 níveis de luminosidade (0% - pleno sol -, 18%, 30%, 50% e 80% de sombreamento), totalizando 40 parcelas com 10 plantas cada. Os dados obtidos foram submetidos à análise de variância e regressão. Os resultados de crescimento indicam maior adaptação, de ambos os genótipos, em condições de irradiância alta a moderada. O índice de qualidade de Dickson demonstrou maior capacidade de sobrevivência em condições de campo a níveis altos de irradiância para o genótipo PH 16 e a níveis de irradiância moderada para IPIRANGA 01. Os teores de pigmentos fotossintetizantes de IPIRANGA 01 mostram-se mais elevados na condição de 30% de sombreamento, enquanto que, para PH 16 os maiores teores foram observados em 50% de sombreamento. Ambos os genótipos demonstraram altas taxas de A, Ci, E, A/E, A/Gs e A/Ci, sob elevadas irradiâncias, assim como adaptações anatômicas caulinares e foliares a maiores luminosidades, tais como, maior espessamento do limbo foliar, parênquima paliçádico, esponjoso, limbo foliar, epiderme adaxial e densidade estomática, além de maior densidade e frequência de elementos de vaso e espessura do xilema secundário. Os genótipos PH 16 e IPIRANGA 01 apresentaram uma grande plasticidade em relação aos diferentes níveis de irradiância, no entanto, constatou-se que PH 16 apresentou melhor desempenho sob condições de alta irradiância, como as obtidas nos tratamentos a pleno sol e 18% de sombreamento, enquanto que, IPIRANGA 01 mostrou-se mais adaptado sob sombreamento moderado, a 30% de sombra. / The luminosity emerges as one of the most important abiotic factors in the establishment of plants and can cause changes in physiological and anatomical, interfering directly in their development and consequently their productivity. The cocoa tree (Theobroma cacao - Malvaceae ) has great economic interest due to the use of its seeds to produce cocoa butter and chocolate. In this sense, the objective of this study was to compare physiological and anatomical characteristics of two genotypes of cacao (PH 16 and IPIRANGA 01), subject to different conditions of solar radiation in order to infer about the lighting conditions most favorable to their development. Analyses of growth, pigment content, gas exchange and anatomical stem and leaf. The experimental design was randomized blocks (DBC), with four replications in a factorial 2 x 5 , consisting of two genotypes and 5 brightness levels ( 0 % - full sun - 18 % , 30 % , 50 % and 80% shading), totaling 40 plots with 10 plants each. Data were subjected to analysis of variance and regression. The results indicate higher growth adaptation in both genotypes under conditions of moderate to high irradiance. The quality index Dickson demonstrated greater ability to survive under field conditions with high levels of irradiance for genotype PH 16 and moderate levels of irradiance for IPIRANGA 01. The content of photosynthetic pigments of IPIRANGA 01 show up higher in the condition of 30% shade, while for PH 16 the highest levels were observed in 50% shade . Both genotypes showed high rates of A, C , E, A / E , A / Gs and A / Ci, under high irradiance, as well as stem and leaf anatomical adaptations to higher luminosities, such as greater thickening of the leaf, parenchyma palisade and spongy, leaf blade, adaxial stomatal density, higher density, frequency of vessel elements and thickness of secondary xylem . Genotypes PH 16 and IPIRANGA 01 showed a large plasticity in relation to different levels of irradiance, however, it was found that PH 16 performed better under high irradiance, as obtained from treatments in full sun and 18% shade, while IPIRANGA 01 was more adapted under moderate shading, 30% shading.

Submitted by Maykon Nascimento (maykon.albani@hotmail.com) on 2015-04-29T19:06:00Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) / Approved for entry into archive by Elizabete Silva (elizabete.silva@ufes.br) on 2015-04-30T20:12:01Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) / Made available in DSpace on 2015-04-30T20:12:01Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Dissertação Gustavo Benevides.pdf: 380662 bytes, checksum: e22589abe3e8dc1a64213785a64889e5 (MD5) Previous issue date: 2015-04-29 / FAPES - Fundação de Amparo a Pesquisa do Espírito Santo / O uso de lipídios obtidos a partir da biomassa de microalgas tem sido descrito como uma alternativa promissora para a indústria petro-diesel e envolve etapas como o cultivo de microalgas, separação da biomassa e extração de lipídios. Para viabilizar a produção em larga escala, é necessário selecionar as espécies mais produtivas, diminuir os custos de produção e determinar as condições ideais de cultivo. Os gêneros Chlorella, Desmodesmus e Ankistrodesmus apresentam características favoráveis à produção comercial, tendo sido então selecionada uma espécie de cada no presente trabalho. O objetivo do estudo foi avaliar diferentes condições de cultivo de Ankistrodesmus fusiformis, Chlorella vulgaris e Desmodesmus spinosus visando o aumento da produtividade em biomassa e lipídios totais. As algas foram identificadas e cultivadas em laboratório, em condições controladas de temperatura a 26ºC (±1), aeração por borbulhamento à pressão ambiente e luminosidade fornecida por lâmpadas fluorescentes, com intensidade de 47,25 μmol de fótons m-2.s-1 (3500 lux), fotoperiodo de 12h e pH 7, sob duas concentrações estressantes de nitrato de sódio (0,10 g/L e 0,05g/L). Os cultivos duraram em média 16 dias, sendo as curvas de crescimento construídas com dados de espectrofotometria óptica coletados a cada 48h, e a biomassa obtida ao final do cultivo por centrifugação e liofilização de cada unidade experimental. Para extração dos lipídios totais, foi utilizada a mistura de clorofórmio: metanol (1:2), segundo a metodologia de Bligh & Dyer (1959). Os tratamentos de estresse em D.spinosus resultaram em maior acúmulo lipídico, com aumento de até 149,7%, porém com drástica diminuição do crescimento e biomassa. Em C. vulgaris, nos tratamentos de estresse, verificou-se apenas ligeiro aumento do peso seco e teor de lipídios, não havendo diferença significativa entre os tratamentos e o controle. Da mesma forma, A.fusiformis não mostrou respostas significativas ao estresse pela redução de nitrato de sódio do meio, havendo ligeira diminuição do conteúdo lipídico e aumento do crescimento e biomassa. Com respostas diferentes para cada espécie estudada, evidencia-se a necessidade do conhecimento da fisiologia e autoecologia da cepa a ser cultivada em escala comercial visando à produção de ácidos graxos para fins de biodiesel. / The use of lipids from microalgae biomass has been described as a promising alternative to the petro-diesel industry and involves steps such as microalgae cultivation, biomass separation and lipid extraction. To facilitate the large-scale production, it is necessary to select the most productive species, reduce production costs and determine optimal growing conditions. Chlorella, Desmodesmus and Ankistrodemsus are genus with favorable characteristics for commercial production, being selected then, one species from each one in the present work. The aim of the study was to evaluate different growth conditions in Ankistrodemsus fusiformis, Chlorella vulgaris and Desmodesmus spinosus aiming to enhance the biomass production and total lipid content. The algae were identified and grown in the laboratory under controlled temperature conditions of 26 ºC (±1), aeration by bubbling at ambient pressure and light provided by fluorescent lamps at an intensity of 47,25 μmol photons m-2.s-1 (3500 lux), 12 hours photoperiod and pH 7, under two stressful concentrations of sodium nitrate (0.10 g/L and 0.05 g/L). The cultures lasted an average of 16 days, with growth curves constructed with optical spectrophotometry data collected every 48 hours, and the biomass was obtained at the end of the cultures by centrifugation and lyophilisation of each experimental unit. For extraction of total lipids, were used the mixture chloroform: methanol (1:2) according to Bligh & Dyer (1959) method. The stress treatments in D. spinosus resulted in increased lipid accumulation, an increase of up to 149.7%, although with a drastic decrease in growth and biomass. In C. vulgaris, the stress treatments were found to only slightly increase the dry weight and lipid content, with no significant difference between the treatments and the control. Similarly, A. fusiformis showed no significant responses to stress by reduction of sodium nitrate in the medium, with a slight decrease of lipid content and increased growth and biomass. With different answers for each studied species, we highlight the need for knowledge of both the physiology and autecology of the strain to be grown on a commercial scale aimed at the production of fatty acids for biodiesel purposes.