In this work particular attention was given to the study of secondary metabolites produced by some plants belonging to the Amaryllidaceae family, in the specific case isoquinoline alkaloids. At the first instance were characterized both qualitatively and quantitatively three different plants belonging to Amaryllidaceae family, such as: Crinum angustum Steud., Pancratium illyricum L., and Leucojum nicaeense Ard. The alkaloids extracts obtained were separately tested against enzymes involved in specific diseases or liable in multifactorial pathologies, like: MMPs, AChE,and PPO. From leaves extract of P.illyricum was isolated a new compound, 11α-hydroxy-O-methylleucotamine, with important role in AChE inbition. Considering the protection role against external bodies carried out by these metabolites in plant, extracts were also assayed against ATCC microorganisms and clinical isolates. Plants with promising pharmacological activities have been the basis for development of in vitro plant models.

This study deals with the discovery and characterization of EXN6 and EXN11 as novel tumor-associated proteins. EXN6 is mainly present in breast and ovary cancers (40 and 35%) while EXN11 is mainly detected in primary and metastatic colon cancer (40%). A characterization of the two proteins confirmed that they could be novel targets for cancer therapy.

Introduzione. Le cellule mesenchimali derivate dal tessuto adiposo (hASC) rappresentano un importante strumento per la terapia cellulare, in quanto derivano da un tessuto adulto abbondante e facilmente reperibile. Con il dispositivo medico Lipogems l’isolamento di tali cellule è eseguito esclusivamente mediante sollecitazioni meccaniche. Il prodotto ottenuto è quindi minimamente manipolato e subito utilizzabile. Ad oggi, il condizionamento pro-differenziativo delle staminali è per lo più attuato mediante molecole di sintesi. Tuttavia, altri fattori possono modulare la fisiologia cellulare, come gli stimoli fisici e molecole naturali. Onde elettromagnetiche hanno indotto in modelli cellulari staminali l’espressione di alcuni marcatori di differenziamento e, in cellule adulte, una riprogrammazione, mentre estratti embrionali di Zebrafish sono risultati antiproliferativi sia in vitro che in vivo. Metodi. La ricerca di nuove strategie differenziative sia di natura fisica che molecolare, nel particolare onde acustiche ed estratti embrionali di Zebrafish, è stata condotta utilizzando come modello cellulare le hASC isolate con Lipogems. Onde acustiche sono state somministrate mediante l’utilizzo di due apparati di trasduzione, un generatore di onde meccaniche e il Cell Exciter . I trattamenti con gli estratti embrionali sono stati effettuati utilizzando diverse concentrazioni e diversi tempi sperimentali. Gli effetti sull’espressione dei marcatori di staminalità e differenziamento relativi ai trattamenti sono stati saggiati in RT-PCR quantitativa relativa e/o in qPCR. Per i trattamenti di tipo molecolare è stata valutata anche la proliferazione. Risultati e conclusioni. La meta-analisi dei dati delle colture di controllo mostra la stabilità d’espressione genica del modello. I trattamenti con i suoni inducono variazioni dell’espressione genica, suggerendo un ruolo regolatorio di tali stimoli, in particolare del processo di commitment cardiovascolare. Due degli estratti embrionali di Zebrafish testati inibiscono la proliferazione alle 72 ore dalla somministrazione. L’analisi d’espressione associata ai trattamenti antiproliferativi suggerisce che tale effetto abbia basi molecolari simili ai processi di differenziamento. / Background. Adipose derived mesenchymal stem cells (hASC) represent an interesting tool for cell therapy, due to their tissue derivation that is abundant and easily available. Using Lipogems, a medical device, stem cells isolation is performed only with mild mechanical forces, giving a minimally manipulated and ready to use fat product. To date, the differentiation processes in vitro are mostly obtained using chemical compounds. Anyway, other factors are shown to be modulators in cell physiology, as physical stimuli and some natural molecules. For instance, electromagnetic waves induced the expression of differentiation markers in stem cell models and adult cells were reprogrammed after treatment, while Zebrafish embryo derived extracts were shown to be antiproliferative both in vitro and in vivo. Methods. The search for new differentiation strategies was focused both on physical waves and Zebrafish extracts, using hASC isolated with Lipogems as cell model. Acoustic waves were gave using two types of transducer, the mechanical wave generator and the Cell Exciter. Zebrafish extracts treatments were performed using different doses and experimental times. Gene expression analysis of stemness and differentiation genes was conducted in both the experimental approaches using quantitative relative RT-PCR and/or qPCR. Proliferation assay was also performed for treatments with natural compounds. Results and conclusions. Control cell culture data meta-analysis revealed that hASC has a stable gene expression in basal conditions. Acoustic waves induced gene expression changes in hASC, in particular in cardiovascular commitment genes, suggesting a regulatory role in differentiation processes. Two of the Zebrafish extracts are shown to inhibit hASC proliferation, with statistical significance after 72 hours from the treatment. Gene espression analysis suggested that this effect could share the same molecular mechanism with differentiation events.

This PhD thesis is focused on the study of the molecular variability of some specific proteins, part of the outer membrane of the pathogen Neisseria meningitidis, and described as protective antigens and important virulence factors. These antigens have been employed as components of the vaccine developed by Novartis Vaccines against N. meningitidis of serogroup B, and their variability in the meningococcal population is a key aspect when the effect of the vaccine is evaluated. The PhD project has led to complete three major studies described in three different manuscritps, of which two have been published and the third is in preparation. The thesis is structured in three main chapters, each of them dedicated to the three studies. The first, described in Chapter 1, is specifically dedicated to the analysis of the molecular conservation of meningococcal antigens in the genomes of all species classified in the genus Neisseria (Conservation of Meningococcal Antigens in the Genus Neisseria. A. Muzzi et al.. 2013. mBio 4 (3)). The second study, described in Chapter 2, focuses on the analysis of the presence and conservation of the antigens in a panel of bacterial isolates obtained from cases of the disease and from healthy individuals, and collected in the same year and in the same geographical area (Conservation of fHbp, NadA, and NHBA in carrier and pathogenic isolates of Neisseria meningitidis collected in the Czech Republic in 1993. A. Muzzi et al.. Manuscript in preparation). Finally, Chapter 3 describes the molecular features of the antigens in a panel of bacterial isolates collected over a period of 50 years, and representatives of the epidemiological history of meningococcal disease in the Netherlands (An Analysis of the Sequence Variability of Meningococcal fHbp, NadA and NHBA over a 50-Year Period in the Netherlands. S. Bambini et al.. 2013. PloS one e65043).

La Inmunodeficiencia Variable Común es un síndrome heterogéneo caracterizado por hipogammaglobulinemia e infecciones recurrentes. La causa no se conoce, aunque se ha propuesto un fallo en la diferenciación final del linfocito B hacia célula de memoria o célula plasmática productora de anticuerpos. Los pacientes pueden clasificarse en 3 grupos (MB0, MB1 o MB2), con distinta presentación y evolución clínica, en función del grado de alteración de los linfocitos B de memoria. Estudiamos si distintos procesos esenciales para la diferenciación del linfocito B están alterados en los pacientes. Para ello, evaluamos: (i) la activación (expresión de CD86 y proliferación celular) en linfocitos B activados con estímulos T-independientes: ODN (ligando de TLR-9) o extractos bacterianos, con o sin anti-IgM (activación a través del BCR); (ii) la diferenciación (expresión de CD38) y producción de IgG, IgA e IgM en linfocitos B activados con anti-CD40 e IL-21 (estímulo T-dependiente) u ODN, en presencia o ausencia de anti-IgM; y (iii) la apoptosis en linfocitos B vírgenes y de memoria activados con anti-CD40, ODN o anti-IgM, en presencia o ausencia de IL-21. Los linfocitos B de pacientes presentan defectos de activación frente a estímulos T-independientes. Su diferenciación hacia células plasmáticas CD38+ es deficiente, y la producción de anticuerpos está disminuida frente a todos los estímulos. Únicamente los linfocitos B de memoria de pacientes MB0 son menos sensibles al rescate de la apoptosis tras activación. Estos resultados identifican defectos de diferenciación del linfocito B que ayudan a explicar la hipogammaglobulinemia y la variabilidad fenotípica de los pacientes, y señalan la necesidad de su correcta clasificación. / Common Variable Immunodeficiency is a heterogeneous syndrome characterized by hypogammaglobulinemia and recurrent infections. The cause of the disease is unknown, although a failure on final B lymphocyte differentiation into memory B lymphocyte or antibody-secreting plasma cell has been suggested. According to the degree of alteration of their memory B lymphocytes compartment, patients are classified into 3 groups (MB0, MB1 and MB2), with different clinical presentation and evolution. We studied whether several essential steps on B lymphocyte differentiation are altered in patients. With this purpose, we evaluated: (i) activation (CD86 expression and cellular proliferation) on B lymphocytes activated with T-independent stimuli: ODN (TLR-9 ligand) or bacterial extracts, with or without anti-IgM (activation through BCR); (ii) differentiation (CD38 expression) and IgG, IgA and IgM production on B lymphocytes activated with anti-CD40 plus IL-21 (T-dependent stimulus) or ODN, in the presence or absence of anti-IgM; and (iii) apoptosis on naïve and memory B lymphocytes activated with anti-CD40, ODN or anti-IgM, in the presence or absence of IL-21. B lymphocytes from patients show decreased activation in response to T-independent stimuli. Their differentiation into CD38+ plasma cells is deficient, and antibodies production is diminished in response to all stimuli. Only memory B lymphocytes from MB0 patients are less sensitive to activation-induced rescue from apoptosis. These results identify B lymphocyte differentiation defects that contribute to explaining the hypogammaglobulinemia and phenotypic variability of patients, and highlight the need to correctly classify them.

No description available.

Las plataformas Micro / Nanofluídicas, simples y miniaturizadas son especialmente interesantes debido a sus ventajas como la reducción de los volúmenes de muestra y reactivos, la disminución del tiempo de análisis, la posibilidad de portabilidad y la integración de técnicas analíticas convencionales. Además, es importante señalar el papel que pueden jugar los nanomateriales en términos de mejora de las propiedades electroquímicas después de ser integrados en plataformas microfluídicas, o incluso modificaciones superficiales de los transductores. Así, la combinación de la nanotecnología, la electroquímica y la microfluícia, podría proporcionar una plataforma de detección muy potente, por lo que, en esta Tesis se estudian diferentes dispositivos microfluídicos con transductores electroquímicos integrados para aplicaciones bioanalíticas. En esta Tesis se exponen también los aspectos generales y los resultados experimentales, a partir de una introducción general, la cual presenta los trabajos más recientes relacionados con el uso de nanomateriales y tecnologías lab-on-a-chip como una sinergia prometedora para una amplia gama de aplicaciones. Después se presenta la detección electroquímica de proteínas mediante el uso de puntos cuánticos como marcadores. En primera instancia, se describe un chip microfluídico híbrido compuesto por una canal de polidimetilsiloxano flexible (PDMS) y policarbonato (PC) como substrato. Este substrato a su vez tiene impreso electrodos serigrafiados integrados de carbono (SCPE). El dispositivo desarrollado combina las ventajas de los chips microfluídicos flexibles, tales como su bajo coste, la posibilidad de ser desechables y la susceptibilidad de ser producidos en masa con las ventajas de la electroquímica por su facilidad de integración y la posibilidad de ser miniaturizables. En la segunda parte, se realizó la detección electroquímica de puntos cuánticos como marcadores en un ensayo para la determinación de un biomarcador de la enfermedad de Alzheimer: Apolipoproteína E (ApoE). El inmunocomplejo se llevó a cabo mediante el uso de partículas magnéticas tosilactivadas, las que fueron a su vez utilizadas como plataforma de preconcentración de muestra dentro de un canal microfluídico. Debido a la necesidad de lograr límites inferiores de detección en inmunoensayos, en esta Tesis se han propuesto diferentes estrategias para mejorar la sensibilidad de los dispositivos. La primera de ellas es el uso de un campo magnético para inmovilizar una mayor cantidad de partículas magnéticas en una disposición controlable dentro de un canal microfluídico con el fin de obtener una zona de precocentración, donde se lleva a cabo el inmunoensayo. La segunda estrategia que se presenta en esta Tesis es el uso de un sistema de reciclaje de fabricación propia. En esta parte, el incremento de la señal de los puntos cuánticos se demuestra mediante el uso de una bomba peristáltica externa conectada a un chip microfluídico que forma un sistema cerrado. Después de esta demostración, se propuso una micro-bomba peristáltica con válvulas integradas. Todas las etapas de fabricación se optimizaron así como también se desarrolló un software para su control. Por último, el bismuto, que es un material bien conocido para aglomerar los metales pesados, fue usado para aglomerar los puntos cuánticos cuyo núcleo está formado por cadmio II, de esta forma se pudo mejorar la señal electroquímica mediante la reducción de los QDs junto con el Bismuto III. Diferentes optimizaciones fueron hechas usando canales microfluídicos. Adicionalmente, se presentan otras nueva plataforma basada en diamante dopado con boro, transductor utilizado para la determinación electroquímica de la atrazina basado en el desarrollo de un magneto-inmunoensayo. Este inmunoensayo se realizó mediante un ensayo competitivo con peroxidasa de rábano silvestre (HRP) como marcador enzimático y micropartículas magnéticas como plataforma de preconcentración. Otra plataforma propuesta es el transistor orgánico de efecto campo de doble puerta, como transductor para biosensores, desarrollado por la tecnología de inyección de tinta sobre un substrato flexible. Este tipo de transistores orgánicos tiene ventajas importantes para biosensores en términos de coste de fabricación y biocompatibilidad, así como su posibilidad de integración en microcanales. Para demostrar la aplicabilidad de este dispositivo en el campo biológico, se ha funcionalizado su capa externa con un anticuerpo de captura que detecta una proteína modelo sin ningún marcador. Se realiza la fabricación del dispositivo, teniendo en cuenta su estructura, los materiales que lo componen, sus características eléctricas y posibles aplicaciones. Por último, se exponen las conclusiones generales y futuras propuestas. / Simple and miniaturized micro / nanofluidic platforms are especially interesting due to their advantages like the reduction of sample and reagent volumes, the decrease of the analysis time, the possibility of portability and the integration of conventional analytical techniques. Furthermore it is important to point out the role that nanomaterials can play in terms of enhancing electrochemical properties after being integrated into the microfluidic platform or even in the electrode, where the detection event will be performed. Combined together, nanotechnology, electrochemistry and microfluidics could provide a really powerful biosensor platform, thus in the present Thesis different microfluidic platforms with integrated electrodes as transducers in biosensing applications were evaluated. General aspects and experimental results are exposed, starting from a General Introduction that describes various aspects related with the use of nanomaterials and lab-on-a-chip technologies as a promising synergy for a wide range of applications. The electrochemical detection of proteins (ex. Apolipoprotein-E, ApoE) by using CdS or CdSe@ZnS Quantum Dots (QDs) as labels has been one of the main objectives of this Thesis. The immunocomplex was performed by using tosylactivated magnetic beads as preconcentration platform into the same microfluidic system. Due to the need to achieve a lower limit of detection of the immunoassays, different strategies for electrochemical signal enhancing are proposed. The first one is the use of a magnetic field to immobilize magnetic beads in a controllable way into a microfluidic channel in order to obtain a stable magnetic plug where the immunoassay is performed. The second strategy is the use of a home-made recycling system. In this part, the increasing signal of QDs is demonstrated by using an external peristaltic pump connected to a microfluidic chip forming a loop system. After this demonstration, a micro-peristaltic pump with integrated valves is also proposed. All the fabrication steps have been optimized and the software for sequential control of the valves also has been developed. Finally, bismuth is used as it is a well-known material that agglomerates with heavy metals. We took advantages of this property for improving the electrochemical signal of QDs, due to the cadmium content that QDs have in their core. Optimization of the bismuth concentration has been done in order to achieve the highest signal. This detection has been performed in batch system as well as in microfluidic mode. In addition, another novel platform for electrochemical determination of a pesticide (atrazine) based on magneto-immunoassay using boron-doped diamond (BDD) electrode is presented. BDD electrode has been modified by electroreduction of potassium tetrachloroplatinate (K2PtCl4) in order to grow platinum nanoparticles (Pt-NPs) onto the electrode surface. The immunoassay was based on a direct competitive assay using horseradish peroxidase (HRP) as enzymatic label and magnetic microparticles as preconcentration platform. A flexible organic double gate Bio-Field Effect Transistor (Bio-FET) developed by inkjet technology onto a flexible substrate is also presented. This kind of organic transducers has important advantages for biosensors in terms of fabrication cost and biocompatibility as well as their integration into microchannels. To demonstrate the applicability of this device in the biological field, its functionalization with a capture antibody, in order to detect a model protein in a label-free mode was performed. The device fabrication, its structure, materials composition optimization, electrical characteristics and other functionalities are also discussed. Finally, the general conclusions are exposed including some opinions / recommendations for further continuation of the research in the field.

Aquest treball s’ha realitzat al Laboratori de Referència d’Enzimologia Clínica (LREC) de la Unitat de Bioquímica de Medicina. El LREC és un laboratori de referència acreditat per les normes ISO 17025 i ISO 15195, i forma part de la xarxa internacional de laboratoris de referència del Joint Committee for Traceability in Laboratory Medicine (JCTLM). Un dels requisits dels laboratoris de referència es conèixer la contribució de les diferents fonts d’incertesa a la incertesa de mesura, el que es coneix amb el nom de “compilació de la incertesa”. L’objectiu d’aquest treball és l’estimació de la incertesa de mesura, mitjançant un estudi de compilació de la incertesa, de dos procediments de mesura de referència primaris: - el procediment de referència primari per la mesura de l’enzim γ-glutamil transferasa (GGT), com a exemple de procediment amb una única reacció. - el procediment de referència primari per la mesura de l’enzim creatina cinasa (CK), com a exemple de procediment amb més d’una reacció. El càlcul de la incertesa s’ha realitzat a partir dels documents “Evaluation of measurement data – Guide to the expresion of uncertainty”, més conegut com GUM i desenvolupat pel Joint Committee for Guides in Metrology (JCGM), i “Quantifying uncertainty in analytical measurement”, recomanat per l’Eurachem. Això va implicar fer un estudi exhaustiu dels procediments de mesura de referència, que va consistir en els següents passos: a) definir el mesurant; b) identificar totes les possibles fonts d’incertesa; c) calcular la incertesa estàndard de cada font, mitjançant una avaluació de tipus A o de tipus B, d) calcular els coeficients de sensibilitat, els quals descriuen com varia el resultat del mesurant al produir canvis en cadascuna de les fonts de incertesa; e) calcular la incertesa combinada, tenint en compte la presència o no de fonts correlacionades i f) calcular la incertesa expandida amb un nivell de confiança d’aproximadament el 95 %. El valor de la incertesa de mesura estimada per el procediment de mesura de la GGT és d’un 2,2 % amb un factor de cobertura (k) de 2. De totes les fonts d’incertesa estudiades únicament 8 han contribuït a la incertesa de mesura. Aquestes fonts d’incertesa, ordenades per la importància en la contribució, són: la variabilitat expressada com la imprecisió en condicions intermèdies de mesura amb un 35,6 %, l’interferent glicina amb un 22,8 %, l’exactitud de les absorbàncies amb un 17,1 %, la longitud d’ona amb un 12,4 %, el pH amb un 8,61 %, la linealitat amb un 1,60 %, la fracció de volum de mostra amb un 1,18 %, i la temperatura amb un 0,68 %. La incertesa de mesura estimada del procediment de mesura de la CK és d’1,9 % (k=2). Les principals fonts d’incertesa amb una contribució significativa a la incertesa de mesura són: la variabilitat expressada com la imprecisió en condicions intermèdies de mesura amb un 50,1 %, l’exactitud de les absorbàncies amb un 23,2 %, el pH amb un 22,4 %, la fracció de volum de mostra amb un 2,16 %, la temperatura amb un 1,35 %, la longitud d’ona amb un 0,37 % i la linealitat amb un 0,34 %. / This work has been carried out in the Laboratori de Referència d’Enzimologia Clinica (LREC) from de biochemistry and molecular biology department of the Universitat Autònoma de Barcelona (UAB). The LREC is a reference laboratory that is accredited for the ISO 17025 and ISO 15195 standards, and also it is part of the international network of references laboratories of the Joint Committee for Traceability in Laboratory Medicine (JCTLM). One of the requirements of the reference laboratories is to know the contribution of the different sources of uncertainty to the measurement uncertainty, the so called “uncertainty budget”. The aim of this work is the estimation of the measurement uncertainty, through an uncertainty budget study, of two primary reference measurement procedures: - the primary reference procedure for the measurement of the catalytic concentration of γ-glutamyltransferase (GGT), as an example of procedure with one reaction. - the primary reference procedure for the measurement of catalytic concentration of creatine kinase (CK), as an example of procedure with different reactions. The calculation of the uncertainty has been based on the documents "Evaluation of measurement data - Guide to the expression of uncertainty," which is known as GUM and was developed by the Joint Committee for Guides in Metrology (JCGM), and "Quantifying uncertainty in analytical measurement ", which is recommended by the Eurachem. So for the uncertainty estimation a comprehensive study of the reference measurement procedures was done. This study consisted of the following steps: a) definition of the measurand; b) identification of all possible sources of uncertainty; c) quantification of the standard uncertainty of each potential source of uncertainty identified, with a type A or type B evaluation; d) calculation of sensitivity coefficient, which describe how the value of the measurand varies with changes in each sources of uncertainty; e) calculation of combined uncertainty considering the presence or not of correlated sources of uncertainty, and f) calculation of expanded uncertainty with a level of confidence of approximately 95 %. The value of the measurement uncertainty estimated for the measurement procedure of GGT is 2,2 % with a coverage factor (k) of 2. From all the sources of uncertainty identified only 8 have contributed to the measurement uncertainty. This sources of uncertainty, ordered by importance in the contribution, are the following: the variability expressed as imprecision in intermediated conditions with a 35,6 %, the inhibition by glycine with a 22,8 %, the absorbance accuracy with a 17,1 %, the wavelength adjustment with a 12,4 %, the pH with a 8,61 %, the linearity with a 1,60 %, the sample volume fraction with a 1,18 % and the temperature with a 0,68 %. The measurement uncertainty estimated for the measurement procedure of CK is 1,9 % (k = 2). The main sources of uncertainty with a significant contribution to the measurement uncertainty are: the variability expressed as imprecision in intermediated conditions with a 50,1 %, the absorbance accuracy with a 23,2 %, the pH with a 22,4 %, the sample volume fraction with a 2,16 %, the temperature with a 1,35 %, the wavelength adjustment with a 0,37 % and the linearity with 0,34 %.

Inaugural dissertation, Universität Basel. / Reprinted from Revue Suisse de Zoologie, v. 15, 1907. Includes bibliographical references (p. 566-573).

Thesis (doctoral)--Hockschule zu Stockholm, 1928. / "Sonderabdruck aus Acta zoologica. Bd. 9, 1928." Includes bibliographical references (p. 177-191).